UNIVERSITATEA DE TIINE AGRICOLE I MEDICIN VETERINAR CLUJ-NAPOCA COALA DOCTORAL FACULTATEA DE MEDICIN VETERINAR

LUCIAN ROMEO FRCAL

CONTRIBUIA POLUANILOR ATMOSFERICI (PARTICULE DE BENTONIT) ASUPRA DECLANRII I EVOLUIEI PROCESELOR INFLAMATORII LA NIVEL RESPIRATOR

REZUMAT AL TEZEI DE DOCTORAT

CONDUCATOR STIINTIFIC Prof. univ. dr. ALEXANDRU POP

CLUJ-NAPOCA 2009

Lucian R. Frcal

Rezumat al Tezei de doctorat

REZUMAT
I. STADIUL ACTUAL AL DOMENIUL CERCETRII CUNOTINELOR N

Impactul polurii i calitatea sczut a aerului respirat, asupra sntii oamenilor i animalelor, a fost sugerat prima dat n urma unor studii epidemiologice n care s-au analizat date clinice obinute din zone cu un grad mai mare de poluare a aerului. Spitalizrile, vizitele medicale dese sau episoadele asmatice au fost corelate de-a lungul timpului cu grade crescute de particule solide din aerul atmosferic n diferite localiti urbane. Efectele acute ale expunerii la particule includ iritaii la nivelul mucoasei nazale i a ochilor sau schimbri funcionale respiratorii. Expunerea cronic este asociat cu tuse, salivare abundent i scderea funciei pulmonare. De asemenea, pe lng simptomele prezentate, studiile experimentale au relevat apariia unor reacii inflamatorii la nivelul cilor aeriene, naintea instalrii modificrilor funciei pulmonare (Bernstein si col., 2004). Poluanii din aer deriv din variate surse, dintre care cea mai important este procesul de ardere a diferiilor combustibili. Poluanii din aer pot fi clasificai n funcie de: sursa de provenien, compoziia chimic, mrimea particulelor i modul de eliberare n mediul interior sau n exterior. Particulele poluante aflate n suspensie, denumite PM (particulate matter) sunt clasificate n trei categorii: PM ordinare (cu diametrul de 2.5-10 m) - deriv de la nivelul solului, praf de la nivelul oselelor, de la construcii sau prin agregarea unor particule mici de combustie; PM fine (<2.5 m) i PM ultrafine (<0.1 m), ultimele dou categorii formndu-se n principal n urma arderii combustibililor (Lagorio si col., 2006). Efectele adverse ale particulelor poluante din atmosfer. Numeroase studii epidemiologice au demonstrat asocierea dintre nivele crescute ale particulelor ambientale i gradul morbiditii i a mortalitii, iar studii experimentale i clinice au artat legtura dintre

Lucian R. Frcal

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particule i diferite funcii ale organismului. Valorile crescute ale PM par a fi duntoare n special subpopulailor n vrst, pacienilor cu boli cardiovasculare preexistente i celor diabetici. PM ambientale fine (<2,5 m diametru, notate PM2.5) sunt reprezentate n principal de particulele carbonate derivate din procesele de combustie. n aerul urban, particulele fine i ultrafine (<100 nm) sunt cele mai numeroase dintre toate particulele i reprezint cea mai mare suprafa raportat la mas. Aceast suprafa poate transporta o cantitate mare de substane toxice din aer adsorbite sau condensate cum ar fi compui organici sau diferite metale. Mecanismele exacte prin care acioneaz poluanii producnd efecte adverse asupra sntii sunt foarte complexe i nu sunt cunoscute n totalitate. Unul din mecanismele de aciune este reprezentat de fenomenul prin care speciile reactive de oxigen produc efecte inflamatorii. Modelul ierarhic al stresului oxidativ, care prin creterea secvenial a dozelor de PM duce la un rspuns celular protectiv (adaptativ) dar i unul lezional. Stresul oxidativ este definit ca o depleie a glutationului intracelular, rezultnd o acumulare de glutation oxidat i o scdere a raportului glutation/glutation oxidat. Stresul oxidativ iniiaz reacii redox-senzoriale (protein-kinaza mitogen activat, cascada factorului kB) acestea acionnd sinergic pentru a activa citokinele proinflamatorii, chemochininele i expresia receptorilor de adeziune. Efectele polurii atmosferice asupra infeciei cu RSV. Alte studii pe aceeai tem au examinat rspunsul gazdei i aprarea antimicrobian a organismului n condiiile expunerii la PM sau la emisii de la motoare. Datele arat c instilarea acestor componente pot atenua clearance-ul bacterian efectuat de macrofage la nivelul pulmonului i altereaz rspunsul organismului la alergeni. Lund n considerare infeciile virale, expunerea la DEP (diesel exhaust particles) poate exacerba rspunsul gazdei la infecii i diminua clearace-ul viral pulmonar. La acestea, se adaug faptul c particulele ajunse la nivel pulmonar pot induce apariia de leziuni i dezvoltarea proceselor inflamatorii. Dup expunerea la DEP, gradul inflamaiei pulmonare asociat RSV a crescut n funcie de nivelul expunerii la poluant. Concluzionnd, se poate spune c inhalarea acut de particule a exacerbat procesele inflamatorii pulmonare i leziunile datorate infeciei cu RSV (Harrod si col., 2003; Steerenberg si col., 2003).

Lucian R. Frcal

Rezumat al Tezei de doctorat

II.

CERCETRI PROPRII

Partea de cercetri proprii prin cele 4 mari studii efectuate, este divizat n 6 subcapitole, pentru o mai bun structurare i nelegere a subiectului abordat. Astfel pentru nceput este prezentat pe larg agentul poluant, reprezentat de particulele de bentonit, folosit n toate experimentele. Apoi sunt descrise efectele lezionale pulmonare n urma expunerii prin diferite tehnici a obolanilor de laborator. Urmeaz datele obinute n cadrul experimentelor pe celulele bronhiale i descrierea amnunit tehnicii de obinere a unei culturi de celule primare, cu scopul utilizrii n experimente de toxicologie in vitro. Urmtorul capitol analizeaz datele referitoare la stresul oxidativ produs de ctre particulele de bentonit, att in vivo ct i in vitro. n ultimul capitol sunt prezentate datele obinute n urma expunerii oarecilor de laborator att la bentonit ct i la un virus cu tropism pulmar. La final sunt efectuate discuiile generale privind subiectul tezei din perspectiva rezultatele obinute n cercetrile de laborator i sunt evideniate concluziile finale.

OBIECTIVELE LUCRRII Aceast lucrare a pornit de la ipoteza c poluanii de natur solid care ajung la nivel pulmonar pot influena capacitatea organismului de a rspunde la ali factori din mediu, cum ar fi agenii patogeni infecioi. Scopul acestei lucrri i a experimentelor efectuate a fost acela de a aprofunda i lmuri o parte din efectele pe care particulele poluante din atmosfer le pot avea asupra aparatului respirator sau a ntregului organism. Pentru a se atinge acest scop au fost efectuate o serie de experimente att pe animale de laborator ct i pe culturi celulare. Prima parte a tezei a avut acest scop, elucidarea ctorva din aspectele patologice care se manifest n cazul interveniei particulelor de argil la nivel respirator. n mod natural expunerea la aceste categorii de particule este de tip ocupaional, fiind ntlnit n special n cariere de prelucrare a acestora. Avnd n vedere proprietile fizice i chimice deosebite pe care bentonita le manifest, lucrarea a vrut s lmureasc o
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parte din efectele biologice pe care le poate avea, odat ajuns n contact cu esuturile i celulele de la nivel pulmonar. Astfel, a fost elaborat un studiu pe animale de laborator (obolani) axat pe aspecte histopatologice (prin evaluarea leziunilor la nivelul esutului pulmonar i a celularitii de la nivelul lichidului de lavaj bronhoalveolar) i biochimice (reprezentat de evaluarea unor markeri de stres oxidativ att la nivel tisular pulmonar ct i la nivel sanguin). De asemenea, o alt variabil a experimentului a fost reprezentat de metodele de expunere, fiind folosite trei tehnici i timpi de expunere diferiti. Al doilea studiu a folosit un model de lucru in vitro, fiind utilizat pentru expunerea la bentonit o linie celular cu provenien de la nivelul epiteliului bronhial. Aceste celule (Calu-3) au o serie de proprieti asemntoare cu celulele originale, care le fac pretabile pentru studii de toxicologie in vitro. n acest experiment s-au efectuat teste legate de modificri la nivelul mebranei celulare i a funciei de barier a acestora, citotoxicitate, eliberare de proteine inflamatorii, nivelul unor markeri de stres oxidativ, capacitatea de absorbie celular sau stimularea expresiei unor gene. Al treilea studiu, efectuat de oareci de laborator, a introdus n variabilele experimentale un agent patogen cu tropism pentru aparatul respirator, acesta fiind reprezentat de virusul pneumoniei de la oarece (virus din genul virusurilor sinciiale respiratorii). S-au urmrit n primul rnd aspecte de fiziologie a respiraiei prin nregistrarea unor parametri funcionali respiratori sau capacitatea de absorbie a pulmonului. De asemenea s-au evaluat leziunile microscopice pulmonare, s-a evideniat dispersia viral la nivel tisular prin marcarea fluorescent a antigenelor virale i s-a determinat titrul virusului din pulmon. Cercetrile s-au desfurat n cadrul urmtoarelor universiti sau institute de cercetri: Universitatea de tiine Agricole i Medicin Veterinar din Cluj-Napoca, Romania: elaborarea metodologiei, evaluarea histopatologic a esutului pulmonar Universitatea de Medicin i Farmacie Iuliu Haieganu din Cluj-Napoca, Romnia: efectuarea expunerii la bentonit i efectuarea testelor de stres oxidativ la animale

Lucian R. Frcal

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Universitatea din Liege, Belgia: efectuarea studiului experimental pe oareci expui la bentonit i infectai cu virusul PVM Institutul de cercetri a Comisiei Europene Joint Research Centre din Ispra, Italia: efectuarea studiului experimental in vitro

PREZENTAREA PARTICULELOR DE BENTONIT FOLOSITE N EXPERIMENTE Bentonita este o argil impur, un silicat de aluminiu absorbant, constituit n mare parte din montmorilonit. Exist dou tipuri de bentonit: sodic i calcic. Ambele tipuri sunt formate din cenu vulcanic, cel mai adesea n prezena apei. Montmorilonita este un mineral silicat de aluminiu care se prezint cel mai frecvent din cristale microscopice, formnd macroscopic diferite argile, fiind componenta principal a bentonitei. Particulele sunt n general de form plat cu un diametru de aproximativ 1 m, grosimea acestora fiind foarte mic (1 nm).

Fig.1. Compoziia chimic a bentonitei folosite n experimente

Din punct de vedere chimic este un hidroxid silicat de sodiu, calciu, aluminiu i magneziu: (Na,Ca)x(Al,Mg)2(Si4O10)(OH)2nH2O. Alte componente frecvent ntlnite sunt potasiul, fierul, concentraia acestora variind n funcie de sursa montmorilonitei. Compoziia utilizat n cadrul experimentelor este preyentat n Fig.1.
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EVALURI HISTOPATOLOGICE PULMONARE I A LAVAJULUI BRONHOALVEOLAR LA ANIMALE DE LABORATOR DUP EXPUNEREA RESPIRATORIE LA BENTONIT Cercetarea de fa, i-a propus evaluarea unor efecte locale pulmonare dar i generale serice care intervin n urma expunerii la un agent poluant solid. Studiul a fost realizat pe obolani (linia Wistar, masculi, cu masa corporal medie de 130g). Metodele de expunere au fost: intranazal, intratraheal i expunere prin ventilare. Expunerea intranazal presupune anestezia animalului (ketamin i xilazin, ip), poziionarea vertical i depunerea sub form de pictur a suspensiei cu particule, la nivelul narinelor (500g/animal n 100L ser fiziologic). Expunerea intratraheal se realizeaz cu ajutorul unei micropipete cu care se ajunge la nivelul primelor inele traheale, folosind aceeai cantitate de pulberi i aceeai anestezie. Expunerea prin ventilare presupune folosirea unei camere speciale n care pulberea este recirculat continuu, expunndu-se ntreg corpul animalului la poluant. Evaluarea efectelor produse de poluant asupra organismului s-a realizat prin examenul lichidului de lavaj bronhoalveolar, examenul histopatologic al esutului pulmonar i analize biochimice sanguine i tisulare. Rezultate Examenul clinic al animalelor pe parcursul experimentului nu a artat modificri semnificative n ceea ce privete starea clinic a animalelor, a greutii corporale sau a comportamentului. La examenul histopatologic al esutului pulmonar, au fost apreciate: gradul leziunilor de la nivel pulmonar, prezena diferitelor tipuri celulare la nivel alveolar i gradul de ncrcare a macrofagelor alveolare cu particule de bentonit. Examenul histopatologic al esutului pulmonar a evideniat la animalele expuse unic la pulberi de bentonit, indiferent de calea de administrare (intranazal sau intratraheal) prezena de leziuni de pneumonie interstiial difuz, congestia septelor i ngroarea acestora, macrofagele alveolare i celule gigante avnd incluzii intracitoplasmatice (Fig.2). La animalele expuse o perioad mai mare la pulberi (prin instilare intranazal sau prin ventilare) leziunile au

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fost mult mai pronunate comparativ cu cele ntlnite la animalele expuse o singur dat la pulberi.

Fig.2. Lot I Pneumonie interstiial difuz; x200

La nivelul lichidului de lavaj bronhoalveolar (Fig.3) se observ procentul majoritar reprezentat de macrofagele alveolare incluznd aici i celulele gigante (bi- sau trinucleate). Celulele epiteliale de la nivelul cilor respiratorii sunt de asemenea n procent semnificativ (30%). Celulele polimorfonucleare sunt reprezentate ntr-un procent de aproximativ 10%. Restul de celule (10%) sunt reprezentate n general de hematii, celule care nu pot fi caracterizate sau celule alterate.

Fig.3. Reprezentarea grafic a tipurilor de celule din lichidul de lavaj bronhoalveolar

Expunerea un timp mai ndelungat a dus la apariia la nivel bronhoalveolar a unui numr mai mare de macrofage alveolare cu rol n
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fagocitatea particulelor strine comparativ cu animalele expuse o singur dat. La animalele expuse cronic se remarc i creterea semnificativ a procentului celulelor gigante. Procentul neutrofilelor a fosr sczut la toate loturile.Celulele epiteliale s-au ncadrat n procente relativ asemntoare la toate loturile (30%), diferene observndu-se la lotul expus IT, metoda fiind mai agresiv. Expunerea repetat a dus la apariia la nivel bronhoalveolar a unui numr mai mare de macrofage alveolare cu rol n fagocitatea particulelor strine comparativ cu animalele expuse o singur dat.

EFECTE BIOLOGICE ALE BENTONITEI IN VITRO, ASUPRA CELULELOR EPITELIALE BRONHIALE Pentru acest model experimental s-a folosit o linie celular uman (Calu-3), de la nivel bronhial, celule care pstreaz multe proprieti ale esutului de origine, cele mai importante fiind funciile de barier i de secreie. Particulele de bentonit, au fost aplicate la nivelul apical al celulelor, fiind suspendate ntr-un mediu lichid (mediu de cultur sau PBS) i supuse n prealabil sonicrii pentru omogenizare. Expunerea s-a efectuat pe o perioad de 1-72 ore, folosind concentraii ntre 1-300 g per cm2. Metodele de investigaie: Funcia barierei epiteliale Rezistena electric trans-epitelial (TEER) Alamar Blue MTT Neutral Red (NRU) LDH SRO (specii reactive de oxigen) MDA (malondialdehida) NO (oxidul nitric) Interleukina IL-8 MUC5, HSP70, HMOX1, MT1X, Radiomarcarea particulelor cu 57Co2+
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Citotoxicitate i viabilitate

Stres oxidativ Producia de citokine Reglarea unor gene Absorbia celular

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Rezultate Expunerea la particulele de bentonit nu a influenat funcia de barier a celulelor Calu-3, dup o expunere de maxim 72 ore. Particulele de bentonit au redus uor viabilitatea celulelor (Fig.4), la concentraii mai mari de 100g/cm2, fiind imposibil de stabilit indicele IC50 (Concentraia de inhibiie 50).

Fig.4. Viabilitatea celular msurat prin testul MTT (% comparativ cu celulele de control) la 48h dup expunerea la bentonit

Datorit proprietii adsorbtive foarte mari a bentonitei, unele protocoluri de lucru au fost influenate, rezultatele fiind foarte dificil de interpretat: IL-8, viabilitatea celular (NRU, LDH). Expresia genelor verificate a fost influenat n sensul creterii moderate, att comparativ ntre concentraiile bentonitei ct i n funcie de timpul de expunere (24, 48 sau 72 ore), ceea ce presupune o activare a sitemelor de aprare celular i iniierea procesului inflamator. Nu s-a observat o absorbie la nivelul celulelor epiteliale, rezultatele fiind negative n urma msurrii bentonitei radiomarcate cu Co57, la nivel intracelular, dup 24 de ore de la expunere. Cele mai relevante i facile tehnici pentru investigarea efectelor toxice ale particulelor de bentonit asupra celulelor Calu-3, au fost: MTT, Alamar blue, TEER i expresia genelor.

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EFECTELE BENTONITEI ASUPRA PROCESELOR OXIDATIVE IN VIVO SI IN VITRO In vivo: Fenomenele de stres oxidativ (lipoperoxidare, oxidarea proteinelor sau activitatea antioxidant local) au fost mai intense la nivel pulmonar comparativ cu valorile nregistrate din plasma sanguin. Markerii stresului oxidativ din plasm au avut valori mai ridicate n cazul expunerii repetate comparativ cu expunerile unice. Oxidarea proteinelor serice a fost mult mai accentuat n urma expunerii repetate iar markerii stresului oxidativ de la nivelul esutului pulmonar au nregistrat valori mai ridicate n cazul expunerilor unice. Mecanismele antioxidante la nivel pulmonar au fost intensificate la animalele expuse unic la pulberi comparativ cu cele expuse repetat. In vitro: Producia speciilor reactive de oxigen este intesificat de expunerea la particulele de bentonit: la un timp de contact de maxim 24 ore creterea SRO este favorizat de concentraii mai mari de 100g/cm2 (Fig.5); La un timp de contact de 48h sau 72 h, se constat o intensificare uoar a produciei RSO i la concentraii mai mici de 100g/cm2, peste aceast concentraie, reacia fiind foarte concludent. Expunerea la bentonit a celulelor bronhiale nu au determinat modificri n ceea ce privete peroxidarea lipidic sau producerea de radicali liberi ai azotului.
300
ROS; H2DCFDA 20M

250 ROS (% control) 200 150 100 50 0 T0 Ctr + 100g/cm2 H2O2 500M 300g/cm2 2h H2O2 1mM CdCl 50M 6h 5g/cm2 CdCl 100M 24h 10g/cm2

Fig.5. Nivelul SRO (%) comparativ la celulele expuse la bentonit i celulele control negative sau control pozitive, dup o expunere de maxim 24 ore

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OBINEREA UNEI BRONHIALE DE LA PORC

CULTURI

CELULARE

PRIMARE

Studiul experimental a avut ca scop obinea unei culturi celulare primare pornind de la epiteliul respirator traheal i bronhial. Tehnica const n efectuarea urmtorilor pai: prelevarea fragmentelor de trahee i bronhii de la porc, igienizarea mecanic a probelor, imersarea n soluie proteolitic (dezagregarea enzimatic), prelevarea propriu-zis a celulelor epiteliale i cultivarea acestora. Tehnici diferite au fost folosite la metoda de digestie enzimatic i la manoperele care au urmat pn la cultivare. S-au obinut rezultate diferite n cazul schimbrii unor pai ai tehnicii de recoltare sau cultivare, precum i datorate compoziiei mediului de cultur. Cele mai bune rezultate s-au constatat n urma folosirii unui mediu cu FCS (10% i 2%) pentru stadiul de aderare celular i multiplicare n primele 2 zile i a utilizrii compusului Ultroser G pentru faza de multiplicare celular ncepnd cu ziua a 3-a. Cultura primar astfel obinut a putut fi folosit n bune condiii i pentru alte metode de laborator (Fig.6).

Fig.6. Celulele epiteliale dup efectuarea reaciei de imunofluorescen (antimouse 568) cu marcarea unei proteine citoplasmatice (Mx1); microscop optic cu UV, x1000

EFECTELE BENTONITEI ASUPRA SEVERITII INFECIEI CU VIRUSUL PNEUMONIEI (SINCIIAL) LA OARECI Experimentul s-a realizat cu scopul evidenierii efectului pe care particulele de bentonit inhalate l pot avea asupra instalrii, evoluiei i a severitii infeciei virale pulmonare cu PVM la oarece.

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Infecia cu virusul pneumoniei oarecilor (PVM) este primul model de infecie viral experimental, realizat pe oarece care are caracteristicile bolii severe de la om produs de virusul sinciial respirator (RSV). PVM este un virus ncapsulat, monocatenar nesegmentat, ARN virus cu un genom de aproximativ 15 kb, aparinnd aceleiai familii (Paramyxoviridae), subfamilii (Pneumovirinae) i gen (Pneumovirus) ca i RSV uman (Domachowske si col., 2004; Rosenberg si col., 2005). Avantajele certe ale utilizrii PVM includ urmtoarele: tablou clinic morbiditate mimeaz constant aspectele observate la copii cu microbronite asociate cu RSV; granulocitoz sever i infiltraie eozinofilic care sunt asemntoare cu modificrile patologice observate la om; evident i masiv replicare a virusului la nivelul esutului pulmonar; evoluia clar a infeciei spre ARDS (Adult Respiratory Distress Syndrome) observat la aproximativ 3% dintre copii cu microbronite cauzate de RSV. n general, infecia cu virusul pneumoniei cauzeaz la nivel epitelial leziuni degenerative (decilieri, necroz i exfolieri) i infiltraii granulocitare/monocitare, cu o foarte uoar hiperplazie epitelial. Nu s-au observat diferene semnificative ntre liniile de oareci, n ceea ce privete leziunile de la nivelul corneilor nazali sau a traheei. n schimb, leziunile de la nivel pulmonar au fost clar diferite n funcie de linia de animale folosit, confirmnd astfel rezultatele obinute la evaluarea funciei respiratorii, modificrile timpilor respiratori nefiind datorate cilor aeriene superioare (Anh si col., 2006; Faisca si col., 2006). PVM (linia J3666) a fost iniial pasat pe oareci BALB/c iar apoi pe linia celular BS-C-1 (epiteliu renal), pentru a realiza o soluie viral stoc. Aceasta a fost diluat 105 n mediu de cultur MEM i pstrat la temperature de 80C, n tuburi cu 1000L/tub. fiind pregtit pentru inoculare la animalele de experiment. Pentru controlul titrului s-au efectuat verificri selective, prin cultivare pe celule BS-C-1, rezultnd valori de ~3 x 107 PFU/mL. Metoda de infectare a constat n instilarea lent a 50 L suspensie viral (1000 PFU/animal), la nivelul narinelor, animalul fiind meninut n poziie vertical pn la inhalarea ntregii cantiti. Evaluarea funciei respiratorii. Tehnica pletismografiei toraco-abdominale utilizeaz pletismograful pentru ntregul corp, format din dou camere (two-chambered, whole body plethysmograph),
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aparat adaptat special pentru oareci. Procedurile tehnice, controlul calitii, procesarea datelor i analiza curbei respiraiei au fost n prealabil validate n cadrul laboratorului. Cu ajutorul pletismografului s-au msurat direct o serie de parametri fiziologici respiratori, pe baza curbei fluxului toracoabdominal: durata inspiraiei (TI-time of inspiration), durata expiraiei (TE-time of expiration), frecvena respiratorie (breathing frequency-FR), volumul total al aerului inspirat si expirat la fiecare respiratie (TV-tidal volume), timpul de relaxare (relaxation time-RT), punctul maxim inspirator (peak inspiratory flow-PIF), punctul maxim expirator (peak expiratory flow-PEF), pauza inspiratorie final (end inspiratory pause-EIP), pauza expiratorie final (end expiratory pauseEEP), timpul necesar pentru expirarea primelor 30% din TV (TE30%), timpul necesar pentru a expira ultimele 5% din TV (TE5%), diferena dintre fluxul respirator nazal i cel toracoabdominal (dT) pe baza creia se calculeaz rezistena specific a cilor aeriene: sRaw = [(TI + TE)/(2 x ) x (Patm 47) x 1.36 x 2 x x dT/(TI + TE)], unde Patm reprezint presiunea atmosferic. Pe baza datelor obinute se pot calcula urmtorii indici: rata respiratorie (the respiratory rate) [RR = 60/(TI + TE)], valoarea ventilaiei (the minute ventilation) [MV = RR x TV] i ciclul respirator (the duty cycle) [%TI = TI/(TI + TE)] Monoxidul de carbon (CO) absorbit s-a determinat cu ajutorul unui aparat care msoar cantitatea de monoxid de carbon din interiorul unei camerei de expunere (CO uptake monitor). Rezultate Evaluarea clinic. Cele mai semnificative simptome au fost observate n ziua 6 i 7 post-infecie, caracterizndu-se prin: slbire, adinamie, schimbarea aspectului prului, ncetarea efecturii toaletei corporale, apariia de secreii oculare abundente. Nu au aprut cazuri de mortalitate n urma anesteziei iar dup efectuarea expunerii la particule i pn la momentul infectrii cu PVM, nu au fost observate modificri clinice. Toate animalele au supravieuit pn n ziua 7 post-infecie, cnd au fost eutanasiate, indiferent de starea clinic. De menionat este faptul c simptomele prezentate au fost mai evidente la animalele din lotul fr bentonit, doar infectate cu PVM.
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Modificri funcionale la nivelul aparatului respirator. O parte din parametri fiziolozici respiratori msurai prin tehnica pletismografiei toraco-abdominale au nregistrat variaii semnificative ntre cele dou loturi. Acestea sunt reprezentate de: frecvena respiratorie (FR), rata respiratorie (RR), timpul inspirator (TI) i cel expirator (TE), pauza inspiratorie final (EIP) i valorile CO absorbit. Cele mai importante modificri au fost observate n zilele 6 i 7 postinfecie, n celelalte zile post-inhalare sau post-infecie, valorile nregistrate fiind constante la ambele loturi. S-a evideniat o modificare a curbei respiratorii (Fig.7), fenomenul fiind prezent la 66% din lotul infectat cu PVM dar fr inhalare de bentonit, fiind constant i caracteristic, n ultimele zile de manifestare a pneumoniei la oarece. Nici un animal din lotul Bent+/PMV+ nu a prezentat aceste modificri.
Bent-/PVM+
5

D0

Di+7

Flow (ml/s)

-1

-2

-3

-4

-5 0 151 303 454 Time (ms) 606 757 1021

Fig.7. Curba toracoabdobimal a respiraiei la animalele din lotul Bent-/PMV+, cu modificarea la nivelul timpului expirator

Leziunile microscopice de la nivelul esutului pulmonar au fost caracterizate prin grade diferite de infiltraie interstiial i mai puin de prezena infiltraiei celulare la nivel alveolar. Diferena dintre cele dou categorii de animale experimentale s-a constatat n ceea ce privete prezena celulelor inflamatorii la nivel interstiial care a fost ma vizibil la animalele din lotul Bent-/PVM+. Nivelul inflamaiei a fost caracterizat prin prezena de celule inflamatorii mononucleare, eozinofile i neutrofile. La majoritatea animalelor s-au observat infiltraii perivasculare cu celule inflamatorii mononucleare. Prezena de celule la nivel alveolar a fost rar pentru animalele din lotul Bent+/PVM+ i moderat pentru cele din lotul Bent-/PVM+,
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macrofagele alveolare fiind dominante la acest nivel. S-au mai ntlnit aspecte de necroz celular i edeme, dar ntr-un grad foarte redus. Titrul viral de la nivel pulmonar a fost determinat n PFU (uniti formate n plac). Valoarea obinut s-a raportat la cantitatea de supernatant obinut dup triturarea esutului pulmonar i per gram esut. S-a observat o diferen net ntre cele dou loturi de animale: 3,38x104 PFU/g la lotul Bent-/PVM+, respectiv 1,37x104 PFU/g la lotul Bent+/PVM+. CONCLUZII FINALE Din datele prezentate se poate afirma c particulele de bentonit la doze i timpi de expunere limitate, au avut un efect toxic moderat, fr a determina leziuni microscopice grave; valori uor mai ridicate se observ n cazul expunerii repetate. Celulele bronhiale expuse la particulele de bentonit au nregistrat modificri caracterizate printr iniierea procesului inflamator, dar efectele asupra membranei sau altor componente celulare nu au avut o intensitate att de mare pentru a produce o citotoxicitate important. n acest caz se pot recomanda cercetrile pe alte tipuri de celule de la nivel respirator, de preferat fiind liniile celulare alveolare, culturile primare sau co-culturile. Particulele de bentonit au acionat ca un factor protector n cazul suprapunerii unei infecii virale pulmonare, modificrile funcionale, lezionale i virale fiind mult diminuate comparativ cu animalele care nu au fost expuse n prealabil la bentonit. i n acest caz se pot ncerca diferite variante de expunere la particule versus infecie pulmonar, att in vivo ct i in vitro, subiectul referitor la aciunea factorilor poluani asupra organismului i asupra severitii unor afeciuni pulmonare rmnnd de actualitate i cu multe elemente nc neelucidate. Lucrarea a reprezentat un exerciiu foarte util din punct de vedere tehnic, cu folosirea a numeroase metode moderne de laborator, iar din punct de vedere medical cercetrile au adus elemente noi n ceea ce privete influena factorilor de mediu asupra organismului, studiile in vivo i in vitro completnd un tablou general cu date importante pentru domeniile fiziopatologiei i a toxicologiei.
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III.
1.

BIBLIOGRAFIE SELECTAT
Anh, D. B., P. Faisca and D. J. Desmecht (2006). "Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains." Am J Physiol Lung Cell Mol Physiol 291(3): L426-35. Bernstein, J. A., N. Alexis, C. Barnes, I. L. Bernstein, A. Nel, D. Peden, D. Diaz-Sanchez, S. M. Tarlo and P. B. Williams (2004). "Health effects of air pollution." J Allergy Clin Immunol 114(5): 1116-23. Domachowske, J. B., C. A. Bonville and H. F. Rosenberg (2004). "Animal models for studying respiratory syncytial virus infection and its long term effects on lung function." Pediatr Infect Dis J 23(11 Suppl): S228-34. Driscoll, K. E., D. L. Costa, G. Hatch, R. Henderson, G. Oberdorster, H. Salem and R. B. Schlesinger (2000). "Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations." Toxicol Sci 55(1): 24-35. Faisca, P., D. B. Tran Anh, A. Thomas and D. Desmecht (2006). "Suppression of pattern-recognition receptor TLR4 sensing does not alter lung responses to pneumovirus infection." Microbes Infect 8(3): 621-7. Forbes, B. and C. Ehrhardt (2005). "Human respiratory epithelial cell culture for drug delivery applications." Eur J Pharm Biopharm 60(2): 193-205. Geh, S., R. Yucel, R. Duffin, C. Albrecht, P. J. Borm, L. Armbruster, M. Raulf-Heimsoth, T. Bruning, E. Hoffmann, A. W. Rettenmeier and E. Dopp (2006). "Cellular uptake and cytotoxic potential of respirable bentonite particles with different quartz contents and chemical modifications in human lung fibroblasts." Arch Toxicol 80(2): 98-106. Harrod, K. S., R. J. Jaramillo, C. L. Rosenberger, S. Z. Wang, J. A. Berger, J. D. McDonald and M. D. Reed (2003). "Increased susceptibility to RSV infection by exposure to inhaled diesel engine emissions." Am J Respir Cell Mol Biol 28(4): 451-63. Hetland, R. B., M. Refsnes, T. Myran, B. V. Johansen, N. Uthus and P. E. Schwarze (2000). "Mineral and/or metal content as critical determinants of particle-induced release of IL-6 and IL-8 from A549 cells." J Toxicol Environ Health A 60(1): 47-65. Hetland, R. B., P. E. Schwarze, B. V. Johansen, T. Myran, N. Uthus and M. Refsnes (2001). "Silica-induced cytokine release from A549 cells: importance of surface area versus size." Hum Exp Toxicol 20(1): 46-55. Lagorio, S., F. Forastiere, R. Pistelli, I. Iavarone, P. Michelozzi, V. Fano, A. Marconi, G. Ziemacki and B. D. Ostro (2006). "Air pollution and lung function among susceptible adult subjects: a panel study." Environ Health 5: 11. Kaan, P. M. and R. G. Hegele (2003). "Interaction between respiratory syncytial virus and particulate matter in guinea pig alveolar macrophages." Am J Respir Cell Mol Biol 28(6): 697-704.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

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13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Li, X. Y., D. Brown, S. Smith, W. MacNee and K. Donaldson (1999). "Shortterm inflammatory responses following intratracheal instillation of fine and ultrafine carbon black in rats." Inhal Toxicol 11(8): 709-31. Morimoto, Y., H. Nagatomo, M. Hirohashi, T. Oyabu, A. Ogami, H. Yamato, K. Kuroda, Y. Obata, T. Higashi and I. Tanaka (2005). "Expression of clara cell secretory protein in the lungs of rats exposed to crystalline silica in vivo." J Occup Health 47(6): 504-9. Maatta, J., M. Lehto, M. Leino, S. Tillander, R. Haapakoski, M. L. Majuri, H. Wolff, S. Rautio, I. Welling, K. Husgafvel-Pursiainen, K. Savolainen and H. Alenius (2006). "Mechanisms of particle-induced pulmonary inflammation in a mouse model: exposure to wood dust." Toxicol Sci 93(1): 96-104. Rosenberg, H. F., C. A. Bonville, A. J. Easton and J. B. Domachowske (2005). "The pneumonia virus of mice infection model for severe respiratory syncytial virus infection: identifying novel targets for therapeutic intervention." Pharmacol Ther 105(1): 1-6. Saber, A. T., N. R. Jacobsen, J. Bornholdt, S. L. Kjaer, M. Dybdahl, L. Risom, S. Loft, U. Vogel and H. Wallin (2006). "Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor." Part Fibre Toxicol 3: 4. Snipes, M. B., B. A. Muggenburg and D. E. Bice (1983). "Translocation of particles from lung lobes or the peritoneal cavity to regional lymph nodes in beagle dogs." J Toxicol Environ Health 11(4-6): 703-12. Steimer, A., E. Haltner and C. M. Lehr (2005). "Cell culture models of the respiratory tract relevant to pulmonary drug delivery." J Aerosol Med 18(2): 137-82. Steerenberg, P. A., C. E. Withagen, J. A. Dormans, W. J. van Dalen, H. van Loveren and F. R. Casee (2003). "Adjuvant activity of various diesel exhaust and ambient particles in two allergic models." J Toxicol Environ Health A 66(15): 1421-39. Steerenberg, P. A., C. E. Withagen, W. J. van Dalen, J. A. Dormans, F. R. Cassee, S. H. Heisterkamp and H. van Loveren (2004). "Adjuvant activity of ambient particulate matter of different sites, sizes, and seasons in a respiratory allergy mouse model." Toxicol Appl Pharmacol 200(3): 186-200. Stoeger, T., C. Reinhard, S. Takenaka, A. Schroeppel, E. Karg, B. Ritter, J. Heyder and H. Schulz (2006). "Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice." Environ Health Perspect 114(3): 328-33.

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UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE DOCTORAL SCHOOL FACULTY OF VETERINARY MEDICINE

LUCIAN ROMEO FRCAL

CONTRIBUTION OF ATMOSPHERIC POLLUTANTS (BENTONITE PARTICLES) IN PROMOTING AND EVOLUTION OF INFLAMMATORY PROCESSES IN RESPIRATORY SYSTEM

SUMMARY OF PH.D. THESIS

SCIENTIFIC COORDINATOR Prof. univ. dr. ALEXANDRU POP

CLUJ-NAPOCA 2009
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SUMMARY
I. CURRENT STATE OF KNOWLEDGE IN THE AREA OF THE RESEARCH

The impact of environmental pollutants and poor air quality on respiratory infections has been suggested primarily based upon epidemiology studies examining clinical data during episodes of air pollution. Acute effects of particulates exposure include irritation of the nose and eyes, lung function changes, respiratory changes, headache, fatigue and nausea. Chronic exposures are associated with cough, sputum production and lung function decrements. In addition to symptoms, exposure studies in animals have documented a number of profound inflammatory changes in the airways, notably, before changes in pulmonary function can be detected. (Bernstein si col., 2004) Air pollution derives from a variety of sources, of which the combustion of fossil-fuel products is the principal source. Air pollutants can be classified by their source, chemical composition, size, and mode of release into indoor or outdoor environments. Suspended particulate pollutants, designated as ambient particulate matter (PM), are classified into 3 categories. Coarse PM (aerodynamic diameter, 2.5-10 m) is derived from abraded soil, road dust (e.g., brake and tire dust), construction debris, or aggregation of smaller combustion particles, whereas fine (<2.5 m) and ultra fine (<0.1 m) PM is primarily formed during the combustion of fossil-fuel products. The adverse effects of PM on health. Numerous epidemiologic studies have demonstrated an association between elevated levels of ambient particles and morbidity or mortality. High levels of particulate matter (PM) seem to be especially harmful to susceptible sub-populations such as the elderly, patients with preexisting cardiopulmonary diseases, and diabetics. Ambient fine-mode PM (<2.5 m particle diameter, PM2.5) consists mainly of anthropogenic, carbonaceous particles derived from combustion processes. In urban air,

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fine and ultrafine particles (<100 nm) are most numerous among all particles and represent the highest surface area per mass. This surface can carry large amounts of adsorbed or condensed toxic air pollutants such as organic compounds and transition metals. The exact mechanism or mechanisms by which air pollutants cause adverse health effects are complex and not properly understood. One mechanism of action is how reactive oxygen species cause inflammation. The hierarchic oxidative stress model, in which incremental doses of PM sequentially induce protective and injurious cellular responses. Oxidative stress is defined as a depletion of intracellular glutathione, leading to accumulation of oxidized glutathione and a decrease in the glutathione/oxidized glutathione ratio. Oxidative stress initiates redox-sensitive signaling pathways (mitogen-activated protein kinase and the nuclear factor kB cascade), which work synergistically to activate proinflammatory cytokine, chemokine, and adhesion receptor expression through appropriate genetic response elements. The effects of atmospheric pollutants in development of RSV infections. In experimental models, PM from DEE exacerbates the host response to bacterial infections of the lung, likely occurring through mechanisms of altered bacterial clearance. In these studies, whole mixtures of inhaled DEE were used to assess the effect of DEE exposure on the host response to subsequent RSV infection in vivo. Multiple levels of DEE exposure were assessed in a relatively RSVresistant mouse strain at the time of peak viral titers following infection. The DEE exposure concentrations resemble levels ranging from high ambient to high levels occupational settings, thus providing information regarding possible health effects of DEE in human disease. Following exposure to DEE, lung inflammation and pathogenesis to RSV infection were increased concordant with decreased RSV clearance in lung tissues. These studies provide the first evidence of increased lung disease to RSV by prior exposure to DEE in vivo, and suggest a role for ambient DEE in the increased susceptibility to respiratory viral infection. (Harrod si col., 2003; Steerenberg si col., 2003)

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II.

ORIGINAL RESEARCHES

The part of thesis including the original research, represented by 4 experimental studies, is divided into 6 chapters, for a better understanding and structuring of the topic. First is presented the particulates bentonite used as a pollutant agent in all experiments. Then are described the pulmonary lesions following the exposure of laboratory rats, by different techniques. Also, are presented the data obtained in the in vitro experiments and the technique performed to obtain a primary cell culture from pigs bronchial epithelium. The next chapter examines data related to the oxidative stress produced by bentonite particles, both in vivo and in vitro experiments. The last chapter present the data obtained from laboratory mice exposed simultaneously to bentonite and a pulmonary virus. Finally, the general discussions on the research subject, related with all results obtained following the laboratory investigations. At the end the final conclusions are highlighted.

OBJECTIVES This research started from the hypothesis that the respirable particulates influence the ability of lung to respond to other environmental factors such as infectious pathogens. The aim of the research and the experiments was to deepen and elucidate some of the effects of particulates pollutant from the atmosphere on the respiratory system. In order to achieve this goal were performed a series of experiments on laboratory animals and cell cultures. The objective of the first research was to elucidate several pathological aspects in case of lung exposure to clay particles. The natural exposure to these types of particles is considered being occupational and is seen especially in bentonite processing areas. Considering the physical and chemical properties of bentonite the research tried to show some of the biological effects that may occur following the contact with the lung tissues and cells. For this aim, was performed a study on laboratory animals (rats) based on
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histopathological aspects (lesions and exam of cells from bronhoalveolar lavage fluid) and biochemical assays (markers of oxidative stress from lung tissue and blood plasma). Also, another variable of the experiment was represented by the exposure methods, using three techniques and different exposure times. The second study was performed using cell culture model of bronchial epithelium. These cells (Calu-3) have certain properties similar to the original cells, which make them suitable for studies of in vitro toxicology. In this experiment has been carried out tests related to cell membrane damages and barrier function, the cytotoxic effects, release of inflammatory proteins, the level of oxidative stress markers, the cell uptake capacity and gene expression. In the third study, performed on laboratory mice, the experimental variables were represented by a virus with tropism for the respiratory system (pneumonia virus of mouse). Were registered the respiratory pattern functions and absorption capacity of lung. Also, it was evaluated the microscopic pulmonary lesions, the virus dispersion in lung by immunofluorescence and quantification of the viral titer in the lung. The universities or research institutes that had contribution in conducting the experimental researches are: University of Agricultural Sciences and Veterinary Medicine from Cluj-Napoca, Romania: methodology design, histopathological evaluation of lung tissue University of Medicine and Pharmacy Iuliu Haieganu from Cluj-Napoca, Romania: exposure to bentonite and oxidative stress assays in rats University of Liege, Belgium: experimental studies on mice exposed to bentonite and infected with PVM Joint Research Center the research institute of European Commission from Ispra, Italy: in vitro experimental studies

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BENTONITE PARTICULATES USED FOR THE EXPERIMENTS The bentonite is an absorbent aluminium phyllosilicate generally impure clay consisting mostly of montmorillonite. Two types exist: swelling bentonite which is also called sodium bentonite and nonswelling bentonite or calcium bentonite. It forms from weathering of volcanic ash, most often in the presence of water. Montmorillonite is a very soft phyllosilicate mineral that typically forms in microscopic crystals, forming a clay. The particles are plate-shaped with an average diameter of approximately 1 micrometer. The particle thickness is extremely small (~1 nm). It is the main constituent of the volcanic ash weathering product, bentonite. Montmorillonite's water content is variable and it increases greatly in volume when it absorbs water.

Fig.1. Chemical composition of bentonite used in the experiments

Chemically it is hydrated sodium calcium aluminium magnesium silicate hydroxide:(Na,Ca)x(Al,Mg)2(Si4O10)(OH)2nH2O. Potassium, iron, and other cations are common substitutes; the exact ratio of cations varies with source. The composition of bentonite used in the experiments is presented in Fig.1.

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HISTOPATHOLOGICAL AND CYTOLOGICAL EVALUATIONS FOLOWING RESPIRATORY EXPOSURE TO BENTONITE PARTICLES OF LABORATORZ ANIMALS The research objective was to evaluate some exposure methods of laboratory animals to pollutant particles. The experimental study was realized on rats (Wistar strain, males, medium weight 130g). The exposure methods: intranasal, intratracheal instillation and exposure by ventilation. Intranasal instillation consist in slowly instilling 500g / animal in 100L of physiological serum into the nostrils of the anesthetized rat (ketamine and xylazine, ip), maintained in a vertical position. The intratracheal instillation was realized with a micropipette directly to the first tracheal rings using the same quantity as in the previous method and the same anesthesia. In the last methods, we used a special chamber in which the particles are recirculated continuously, exposing the whole body to the pollutant. We evaluated the effects by analyzing the bronchoalveolar lavage liquid, histopathological exam of the lung tissue and biochemical exam of blood and tissue. Results The clinical exam of the animals during the experiment doesnt show significant symptoms due to the bentonite instillation or inhalation. The histopathological lesions observed were characterized by alveolar macrophages and giant cells infiltration. In the cytoplasm of phagocytes were observed particles inclusions, obviously on IT group comparatively with IN group. Also, the lesions were more severe on chronic exposure groups. It was observed thickening of alveolar walls and a large number of alveolar macrophages with bentonite inclusions (Fig.2). The histopathological examination shows on the single expose animals, in the both methods (IN or IT), light inflammatory lesions characterized by cells infiltrations, alveolar macrophages or giant cells. The cytoplasmic inclusions were more evident on the IT/Ac group. On the chronic expose animals, the main observed lesions are the thickening of the alveolar walls and a visible infiltration with inflammatory cells. A long-time exposure leads to an obviously
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development of histopathological lesions comparatively with a single instillation.

Fig.2. Group I Diffuse interstitial pneumonia, x200

In the bronchoalveolar lavage fluid (Fig.3) the alveolar macrophages (AM) are the main cells represented, including here also the bi- or tri-nucleated cells (Gcells). The airway epithelial cells (AEpithCells) are also in a large number (about 30%), the PMN cells percent is about 10% and other cells (red blood cells, uncharacterized cells, etc.). All the experimental samples registered increases of cells number comparatively with the reference group.

Fig.3. Chart of cells type from bronchoalveolar lavage fluid

An increase of AM on the animals chronically exposes to particles (VE and IN/Chr) - comparatively with the single expose groups (IN/Ac and IT/Ac). Also the bi- or tri-nucleated cells were increase on repeated expose animals. The PMN had low values in all groups. The
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epithelial cells were in equal values in the all groups (30%), the exception observed on the IT/Ac group, fact caused by the exposure method, the intratracheal administration being more aggressive. The repeated exposure to bentonite particles leads to an increase of the alveolar macrophages with an active role in particles phagocytosis if we compare with the single expose animals or with the reference animals. Also, the same difference was registered for the multinucleated cells.

BIOLOGIC EFFECTS OF BRONCHIAL EPITHELIUM CELLS

BENTONITE

IN

VITRO,

ON

The experimental model was applied on a human bronchial cell line (Calu-3) and was performed biological assays effects of inhalation pollutants that could affect the airway lung tract or the lung tissue. The general scheme of the experiments consists in: suspension of bentonite particles in PBS or culture medium; Sonication and vortex to homogenize the suspension; Exposure of Calu-3 cells on the apical side (Exposure time 1-72 hours; Concentrations: 1 to 300 g per cm2. The end-points performed: Barrier function TEER (Trans-epithelial electrical resistance) NRU (Neutral Red Uptake) Alamar Blue MTT LDH (Lactate-dehydrogenase) NO (Nitric oxide) ROS (Reactive Oxygen Species) Lipids peroxidation(MDA) IL-8 (Interleukin 8) MUC5, HSP70, HMOX1, MT1X, Radiolabeled particles with 57Co2+

Cytotoxicity

Oxidative stress Cytokines release Gene expression Cellular uptake of bentonite

Results: No effect of bentonite particles on the integrity of the barrier (measured by TEER) after 72 hours of exposure
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Bentonite particles reduced slightly cell viability (Fig.4) only at high concentrations (>100g/cm2)

Fig.4. Cell viability by MTT assay (comparative with negative control cells) after 48h of contact with bentonite

Due to the adsorptive property of bentonite, some of the assays are difficult to perform and the results are difficult to analyze: IL-8, NRU or LDH The genes (MUC5, HSP70, HMOX1, MT1X) were slightly up-regulated comparing different concentration or the exposure time Considering the adsorptive property of bentonite, the methods more reliable for in vitro model of exposure, are: MTT, Alamar blue, oxidative stress assays (ROS and MDA) and genes expression

THE EFFECTS OF BENTONITE ON OXIDATIVE STRESS IN VIVO

AND IN VITRO

In vivo: oxidative stress reactions (lipids and protein oxidation or the antioxidant activity) were more intense in the lung compared with values recorded in the blood plasma. Oxidative stress markers in plasma values were higher when repeated exposure was performed, compared with single exposure. Serum protein oxidation was more pronounced after repeated exposure and the markers of oxidative stress in the lung tissue had higher values for single exposures. Antioxidant mechanisms

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in the lung were increased in animals with single exposure compared to those exposed repeatedly. In vitro: the release of reactive oxygen species was increased by exposure to bentonite particles: for a contact within 24 hours, increasing of ROS was observed only for concentrations higher than 100g/cm2 (Fig.5); for a contact time of 48h and 72 h, was observed a slight intensification of ROS production also for concentrations less than 100g/cm2; above these concentrations, the response is very conclusive. Exposure of bronchial cells to bentonite has no effects in lipid peroxidation or nitrogen free radicals production.
300
ROS; H2DCFDA 20M

250 ROS (% control) 200 150 100 50 0 T0 Ctr + 100g/cm2 H2O2 500M 300g/cm2 2h H2O2 1mM CdCl 50M 6h 5g/cm2 CdCl 100M 24h 10g/cm2

Fig.5. ROS levels (%) for the cells exposed to bentonite, compared with control negative or positive control cells, after an exposure within 24 hours

THE OBTAINING OF A PRYMARY CELL CULTURE FROM PIGS BRONCHIAL EPITHELIUM Primary cell culture from pig The aim of this experimental study is to obtain a primary cell culture from pigs airway epithelium (trachea and bronchia). The technique consists in trachea and bronchi sampling, cleaning the adherent tissues, enzymatic proteolysis, collecting the cells and proper cultivation of epithelial cells. There were applied different techniques for the enzymatic digestion and for the cultivation steps. Different results were observed due to changes of cell digestion method and due
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to the different culture medium composition. The best primary cell culture was obtained with a 10% and 2% of FCS for the adhesion steps respectively for the first two days of multiplication and the replacing of FCS with Ultroser G starting with the 3rd day. Hereby, the primary cell culture was very well used for other laboratory methods (Fig.6).

Fig.6. Immunofluorescence reaction (antimouse 568) for a citoplasmatic protein (Mx1) of epithelial cells,UV optical microscope, x1000

EFFECT OF RESPIRATORY EXPOSURE TO BENTONITE ON THE SEVERITY OF PNEUMOVIRUS PRIMO-INFECTION IN A MOUSE MODEL The objective of this study was to show the eventuality that, the acute exposure to bentonite particles by intranasal instillation has an impact in the evolution / severity of pneumonia of mice, induced by PVM. The infection with pneumonia virus of mice (PVM) is the first model that replicates features of severe human respiratory syncytial virus (hRSV) disease in the mouse. PVM is an enveloped, singlestranded nonsegmented, negative-sense RNA virus belonging to the same family (Paramyxoviridae), subfamily (Pneumovirinae), and genus (Pneumovirus) as hRSV (Domachowske si col., 2004; Rosenberg si col., 2005). The advantages of PVM model include the following: clinical picture-morbidity-consistently mimicking that observed in infants with RSV-associated bronchiolitis; dramatic granulocytic and eosinophilic infiltrations that parallel the pathological changes observed in humans; clear evidence of widespread viral replication in lung tissue; clear progression to ARDS, as reported for 3% of infants with RSV

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bronchiolitis. In general, PVM infection caused epithelial regressive (deciliation, degeneration, necrosis, and exfoliation) lesions and inflammatory granulocytic/mononuclear infiltrations, with very little epithelial hyperplasia. Whereas no obvious between-strain difference was recorded in turbinates and tracheae, lung lesions were clearly strain specific, confirming that the differences recorded in the RPF values are not attributable to the upper airways (Anh si col., 2006; Faisca si col., 2006). PVM strain J3666 was first passed in BALB/c mice and then grown once onto BS-C-1 cells to produce the stock solution. The stock solution was then diluted to 105 in MEM, divided into aliquots, and stored at 80C to serve as inoculums. Randomly selected aliquots yielded highly reproducible titers on BS-C-1 cells, amounting to 3 x 107 PFU/mL. The inoculation procedure consisted of slowly instilling 50 L of the viral suspension (1000 PFU/animal) into the nostrils of the anesthetized mouse maintained in a vertical position (35 mg/kg pentobarbital sodium ip). Examination of respiratory function Respiratory pattern/function (RPF) values were measured with the two-chambered, whole body plethysmograph, with practical procedures, quality controls, raw data processing, and respiratory flow curve analysis that were validated previously in the laboratory. A series of parameters was measured directly on the basis of the thoracoabdominal flow curve: duration of inspiration (TI), duration of expiration (TE), tidal volume (TV), breathing frequency (FR), (PIF), expired volume (EV), relaxation time (RT), end inspiratory pause (EIP) end expiratory pause (EEP), peak expiratory flow (PEF) and peak inspiratory flow (PIF). The delay observed between nasal and thoracoabdominal flows (dT) was measured and used to calculate the specific resistance of the airways: sRaw = [(TI + TE)/(2 x ) x (Patm 47) x 1.36 x 2 x x dT/(TI + TE)] - where Patm is atmospheric pressure. On the basis of the parameters measured above, were calculated: the respiratory rate [RR = 60/(TI + TE)], the minute ventilation (MV = RR x TV) and the duty cycle [%TI = TI/(TI + TE)]. CO uptake values were measured in the same mice with the CO uptake monitor.
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Results Clinical data. The most significant clinical symptoms were observed in the 6th and 7th day post-infection (weakness, changes of hair aspect, increases of eye secretions). The infection was performed 24 hours after the bentonite instillation and in this period of time was not observed clinical effect due to the anesthesia or the bentonite dust. Hundred percent of the animals have survive all the experiment period. Respiratory pattern function. The respiratory parameters that show significant differences between two experimental groups were: the breathing frequency (FR), the respiratory rate (RR), the inspiratory and expiratory times (TI and TE), the duty cycle (%TI), the end inspiratory pause (EIP) and the CO uptake values. The most important changes of these respiratory parameters were observed at the interval 6 and 7 days post-infection, the most statistical significance being registered for FR, RR, TI, EIP and CO Uptake. Also, the thoracoabdominal flow curve registered in the first experimental days and at the 7 days post-infection was different for the two groups. It was registered a lengthening of the respiratory time (Fig.7) at 66% of the Bent-/PVM+ mice. The thoracoabdominal flow curve for the Bent+/PVM+ was stable for all the experimental period inclusive in the 7th day pi.
Bent-/PVM+
5

D0

Di+7

Flow (ml/s)

-1

-2

-3

-4

-5 0 151 303 454 Time (ms) 606 757 1021

Fig.7. Thoracoabdominal respiratory curve, in the animals from Bent-/PMV+ group, with a functional delay in the expiratory time

Histological data. The lung inflammatory lesions were characterized by different degrees of interstitial infiltration and less alveolar cell infiltration in both experimental groups. The differences between groups were observed concerning the presence of inflammatory
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cells in interstitium that was more visible on the Bent-/PVM+ animals. The inflammation at this level was caracterised by presence of the mononucleated inflammatory cells, eosinophils and neutrophils cells. The perivascular infiltration with mononuclear inflammatory cells was observed at the majority animals. The presence of cells in the lung alveoli were rarely for the Bent+/PVM+ animals and moderate for the Bent-/PVM+ animals, the alveolar macrophages being the most important cell type. Other facts observed at this level were the presence of cells necrosis and a very low level of edema. The table below shows the levels of inflammatory lesions for each animal and the average for each group. Virological data. The PFU/mL and the PFU/g lung obtained at the 7 day post-infection are presented in Table 4. It can be observed a difference between the PFU values for the experimental groups (3,38x104 PFU/g for Bent-/PVM+ group, respectively 1,37x104 PFU/g for Bent+/PVM+ group). CONCLUSIONS Analyzing the data presented it can be affirmed that the single exposure to particulate bentonite had a moderate toxic effect without causing severe microscopic lesions, slightly increase values observed in the case of repeated exposure. The reaction of bronchial cells exposed to particles was characterized by an initiation of inflammatory process, but the effects on the membrane or other cellular components have not high intensity to produce significant cytotoxicity. In this case we may suggest performing research on other types of cells from the respiratory system, preferably the alveolar cell lines, primary cultures or co-cultures. Bentonite particles acted as a protective factor for the overlap of a viral pulmonary infection. The functional changes and the pulmonary viral titer were much reduced in the animals exposed to bentonite compared with the animals not exposed to bentonite. Also, in this situation can be suggested for future studies, different variations of exposure to particulate versus pulmonary infection, both in vivo and in vitro, this area of the pollutants effects on the severity of lung disease, having many elements still unclear.
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The research was a useful exercise from a technical point of view, using many modern laboratory methods; in the area of medical research has brought new elements regarding the influence of environmental factors on the respiratory system, both studies in vivo and in vitro completing a general picture with valuable data for pathophysiology and toxicology fields. IV.
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