Ispitivanje genotoksičnosti klorpirifosa, imidakloprida i alfa cipermetrina Komet i Komet-FISH testom na produženim kulturama limfocita čovjeka

master's thesis

• Title (english): Evaluation of genotoxicity of chlorpyrifos, imidaclopramid and alpha cypermethrine using Comet and Comet-FISH assays in extended-term human lymphocytes
• Author: Benjamin Vinković
• Mentor: Maja Šegvić Klarić (mentor)
• Committee member: Maja Šegvić Klarić (predsjednik povjerenstva)
• Committee member: Ana-Marija Domijan (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of microbiology); Zagreb
• Defense date and country: 2016-09-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Tekst sažetka Insekticidi su u današnje vrijeme neizostavan dio uzgoja hrane kojima je kroz tu hranu izložena cjelokupna populacija. Stoga je bitno saznati kakve učinke te tvari imaju na ljudski organizam i zdravlje i odrediti regulativu koja će najbolje izbalansirati moguće štetne posljedice i uporabu insekticida. Jedan od bitnih učinaka je genotoksičnost koja se može pokušati odrediti raznim metodama poput ovdje korištenih komet i komet-FISH testova. Komet test je alkalna elektroforeza DNA pojedinačnih stanica u gelu gdje se oštećenja na molekuli DNA očituju kao 'repovi' a očitavanjem dužine i intenziteta repa možemo saznati opću razinu oštećenja molekule DNA. Stanice korištene za eksperiment su bile stanice limfocita iz čovjeka. Ta metoda se već dugo vremena koristi u određivanju genotoksičnosti tvari, ali iako je dosta osjetljiva govori nam samo o općem stanju cjelokupne molekule DNA. Kako bi se dobio uvid u oštećenja specifičnih gena ili struktura molekule DNA primjenio se komet-FISH test. To je kombinacija komet testa s fluorescentnom in-situ hibridizacijom pri čemu određeni slijed molekule DNA obilježimo fluorescentnom bojom pri čemu dobijemo probu koju možemo hibridizirati s nukleoidom u dobivenom kometu čime možemo vidjeti smještaj i izgled promatranog gena kao fluorescentnog signala u sklopu kometa. Nažalost unatoč mogućnostima koje nudi ova metoda nije korištena u puno istraživanja zbog čega ovaj diplomski rad predstavlja značajan doprinos saznanjima o metodi i genotoksičnom učinku ispitivanih insekticida. Nakon dugotrajnog (14 dana) izlaganja limfocita izabranim insekticidima – klorpirifosa, imidakloprida i alfa cipermetrina – u malim koncentracijama (ADI, REL, OEL) i kontrolnim spojevima, izmjerila se dužina i intenzitet repa kometa te je određen broj delecija i migracija ciljanog gena u kometima. Nije pokazana statistički značajna razlika u vrijednosti komet niti komet-FISH testa nakon tretiranja stanica odabranim insketicidima u usporedbi s kontrolnim supstancama. Razmatranjem dostupnih literaturnih podataka došli smo do zaključka da ukoliko se insekticidi koriste ispravno i u propisanim dozama ne bi trebalo doći do štetnih posljedica.

Abstract (english)

Text In modern times insectides are an integral part of food production through which means the entire population is exposed. Therefore, it’s paramount to determine the effects such substances have on human organism and health so that regulations for usage with best can be made. One important effect that they might cause is genotoxicity which could be assessed by methods such as comet and comet-FISH assays. Comet test is gel electrophoresis of DNA of single cells usually under alkaline conditions in which damaged DNA is observed as tails of comets. General level of overall DNA damage can be determined by measuring tail length and intensity. For the purpose of this experiment cultured human lymphocytes out of peripheral blood were used. While comet assay is relatively sensitive method and has been used for a long time in assessing genotoxicity it provides insight only into general state of damage of the whole DNA. So in order to gain insight into damage of specific genes of DNA structures comet-FISH assay has been used. Comet-FISH is a combination of comet assay with fluorescent in-situ hybridization where a specific sequence of DNA is labelled with a fluorochrome that can be annealed to comet DNA thus allowing us to observe position and appearance of wanted gene as fluorescent signal in comets. Unfortunately despite its many possible applications this method isn’t widely used which makes this study important for future reference and insight into method. After exposing lymphocytes for a long time (14 days) to selected insecticides- chlorpyrifos, imidacloprid and alpha cypermethrine- in low concentrations (ADI, REL, OEL) and control substances, tail length and intensity of comets were measured and the number of deletions and migrations of FISH signals were counted. After statistical analysis no significant values, in comet nor comet-FISH assays, were observed in comparison to control substances. After comparing results obtained by previous studies with ones provided by this study it was concluded that with proper care and usage of these insecticides it should come to no harmful effects.

• Keywords: genotoksičnost; komet; komet-FISH; TP53; insekticidi limfociti
• Keywords (english): genotoxicity; comet; comet-FISH; TP53; insecticides; lymphocytes
• Language: croatian
• URN:NBN: urn:nbn:hr:163:588426
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-12-05 09:16:58