Detection Measurement of double-stranded DNA damage in patients with systemic sclerosis using the Comet Assay

Sistemska skleroza (SSc) je kronična avtoimunska bolezen, za katero je značilno nalaganje kolagena v zunajceličnem matriksu, kar privede do fibrotičnih sprememb kože in notranjih organov. Točen vzrok nastanka bolezni še ni znan, dokazano pa je, da se bolezen razvije pod okoljskimi vplivi ob sočasni genetski predispoziciji, ki pogojuje tudi napredovanje bolezni. Dodatno na razvoj vplivajo še različni patofiziološki procesi, ki so hkrati odgovorni še za heterogeno klinično izražanje bolezni in nastanek specifičnih avtoprotiteles. Pri SSc sta v literaturi opisani povečana genomska nestabilnost in povečano število poškodb DNA, prav tako so pri tako okvarjenih celicah oslabljeni tudi popravljalni mehanizmi. Naš namen je bil preveriti, ali lahko z metodo Comet zaznamo povečane endogene poškodbe dvoverižne DNA pri bolnikih s SSc. Hkrati smo opazovali razliko med nastalo škodo ob izzvani oksidativni poškodbi in sposobnost popravljanja oksidativne poškodbe in vitro v levkocitih bolnikov s SSc v primerjavi z zdravimi kontrolami. Zato smo preverjali obseg poškodb DNA v krvi takoj po odvzemu vzorca, po 30 minutni poškodbi z vodikovim peroksidom in 45 minut po odstranitvi vodikovega peroksida, torej po delovanju popravljalnih mehanizmov DNA. Zaradi enostavnosti izvedbe, velike občutljivosti ter možnosti avtomatizacije smo izbrali metodo Comet, ki nam omogoča oceno obsega poškodb DNA in vitro. Preparate bolnikov s SSc in zdravih kontrol smo pregledovali pod mikroskopom ter jih tako ovrednotili semikvantitativno in avtomatsko s programom OpenComet, ki slike preparatov ovrednoti kvantitativno preko več parametrov. Najpomembnejši za oceno poškodb DNA so parametri za opredelitev značilnosti repa kometa, torej dolžina repa, odstotek DNA v repu, repni moment in olivni moment. Preverjali smo tudi skladnost ročnega in avtomatiziranega pregleda ter povezavo med starostjo, spolom, prisotnostjo specifičnih avtoprotiteles ter številom prelomov dvoverižne DNA in sposobnostjo popravljanja. Ugotovili smo, da semikvantitativno in kvantitativno vrednotenje kometov pri metodi Comet dobro korelirata. Obseg endogenih poškodb dvoverižne DNA, ugotovljen z metodo Comet, se statistično značilno ne razlikuje med celotno skupino bolnikov s SSc in kontrolno skupino zdravih. Količine endogenih poškodb dvoverižne DNA in izzvanih eksogenih poškodb dvoverižne DNA so statistično značilno večje pri starejših kontrolah kot pri zdravih mlajših posameznikih. S starostjo pri kontrolah pade učinkovitost popravljalnih mehanizmov DNA, saj smo pri starejših kontrolah neodvisno od spola dokazali statistično značilno daljši repni moment. Pri bolnikih s SSc ne dokažemo povezav med starostjo in številom endogenih poškodb dvoverižne DNA, prav tako ni statistično značilne povezave med starostjo bolnikov s SSc in številom izzvanih poškodb dvoverižne DNA po nanosu vodikovega peroksida ali po delovanju popravljalnih mehanizmov DNA. Med bolniki s SSc s prisotnimi specifičnimi protitelesi proti topoizomerazi ① in s prisotnimi specifičnimi anti-centromernimi protitelesi smo dokazali statistično značilne razlike v repnem momentu. To kaže na razlike v številu endogenih poškodb dvoverižne DNA, ki jih je več pri skupini bolnikov s SSc s prisotnimi anti-centromernimi protitelesi.Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by accumulation of extracellular matrix components, especially collagen, which leads to fibrosis of the skin and internal organs. SSc shows a complex etiology and the exact cause of the disease is still unknown, however both environmental and genetic factors seem to influence the onset and outcome of the disease. Additionally, different pathophysiological processes influence the clinical heterogeneity of the disease and production of specific autoantibodies. Patients with SSc present with increased genomic instability, elevated DNA damage and impaired DNA repair mechanisms. The aim of our study was to define whether it is possible to detect elevated endogenous double-stranded DNA damage with the Comet assay in patients with SSc. Moreover, we assessed if hydrogen peroxide induces DNA damage and evaluated the efficiency of the DNA repair mechanisms in vitro, in leukocytes of patients with SSc and healthy controls. Therefore, we tested the amount of endogenous DNA damage in blood, immediately after blood sampling, 30 minutes after the application of hydrogen peroxide and 45 minutes after the removal of hydrogen peroxide, when the DNA repair activity already began. The Comet assay is a simple and sensitive technique for the determination of DNA breaks in vitro which enables automation. We evaluated prepared blood samples of patients with SSc and healthy controls in two different ways. Semiquantitative measurement included examination of the comets under the microscope by hand and quantitative measurement consisted of taking the images of the comets and automatically analyzing them with the software tool OpenComet. The programme identifies the comets and measures their parameters, of which the most important are tail parameters (the tail length, the percentage of DNA in the tail, the tail moment and the olive moment). Finally, we determined the compliance of the semiquantitative and quantitative evaluation and possible associations of the amount of DNA breaks and efficiency of the DNA repair mechanisms with age, gender or the presence of specific autoantibodies. We established a good correlation between semiquantitative and quantitative evaluation of the comets in the Comet assay. The amount of endogenous double-stranded DNA damage was not significantly different between patients with SSc and healthy controls. The amount of endogenous double-stranded DNA breaks and the amount of induced exogenous double-stranded DNA breaks were significantly higher in older healthy controls then in younger healthy individuals. Since age decreases the efficiency of the DNA repair mechanisms, we also demonstrated that DNA repair mechanisms were significantly less efficient in older controls, independent of gender, and the tail moment was much more profound. These changes were not observed in patients with SSc, in any of the three treatments. However, when comparing patients with different autoantibodies, we found significantly higher amount of endogenous double-stranded DNA damage in SSc patients with anticentromere antibodies than in SSc patients with anti-topoisomerase ① antibodies, indicating changes in endogenous DNA damage depend on the autoantibodies present