Utjecaj cirkadijalnih gena i metamfetamina na oksidativni status Drosophile melanogaster

Methamphetamine (METH) is a potent psychostimulant that causes relapsing substance addiction. Negative consequences of substance abuse are numerous and challenging to study in humans. Fortunately, effects of addictive drugs can be studied in model organisms, such as Drosophila melanogaster or fruit fly. Fruit flies are ideal for this research because their genome is sequenced and extensively studied. Methamphetamine causes elevated reactive oxygen species (ROS) production and disrupts their elimination, leading to oxidative stress. Circadian genes are partially responsible for ROS cycling and in Drosophila four core circadian genes are: period (per), timeless (tim), cycle (cyc) and clock (Clk). In this paper hydrogen peroxide (H2O2) concentration is followed after two exposures to volatilized METH in heads of five fly genotypes: wild type Canton S., per01, tim01, ClkJrk and cyc01. Hydrogen peroxide concentration was measured with dihydroethidium (DHE) stain. Concentration of H2O2 is expected to increase with higher METH exposure, and differences in H2O2 amount should also be noticed between the genotypes. Results show different H2O2 levels among genotypes and this provides insight into the role of circadian genes in the regulation of the oxidative status of Drosophila. The obtained data shows that wild type, per01 and tim01 flies show decrease with first vMETH dose, followed by increase in H2O2 concentration after the second dose. ClkJrk and cyc01 mutants show steady increase in H2O2 concentration with each dose. This shows that irrespective of the mutation METH leads to the increase in the oxidative species and poses questions about the molecular mechanism involved in the regulation of redox state after psychostimulant exposure.Metamfetamin (METH) je snažan psihostimulans koji uzrokuje relapsirajuću ovisnost. Negativne posljedice ovisnosti o drogama brojne su i izazovno ih je izučavati na ljudima. No, učinci droga mogu se vrlo jednostavno inducirati u modelnim organizmima kao što je Drosophila melanogaster ili vinska mušica. Vinske mušice pogodni su organizam za izučavanje u ovakvim istraživanjima jer su njihovi geni sekvencirani i dobro proučeni. Metamfetamin uzrokuje povišenu proizvodnju reaktivnih kisikovih vrsta (ROS) i remeti njihovo uklanjanje, što dovodi do oksidativnog stresa. Cirkadijalni geni su djelomično odgovorni za kruženje ROS-a, a četiri ključna cirkadijalna gena u Drosophile su: period (per), timeless (tim), cycle (cyc) i clock (Clk). Ovaj rad prati koncentraciju vodikovog peroksida (H2O2) u glavama mušica nakon drugog izlaganja volatiliziranom METH-u. Oscilaciju koncentracija pratilo se u pet genotipa mušica (wild type Canton S., per01, tim01, ClkJrk i cyc01) pomoću dihidroetidija (DHE). U rezultatima se očekuju razlike u koncentraciji među genotipovima i porast koncentracije vodikova peroksida sa porastom broja izlaganja METH-u. Rezultati ukazuju na različite razine vodikova peroksida u različitim genotipovima i to pruža uvid u ulogu cirkadijalnih gena u oksidativnom statusu Drosophile. Nadalje, rezultati prikazuju da u wild type, per01 i tim01 mušica, koncentracija H2O2 pada nakon prve, a raste nakon druge vMETH doze, dok u ClkJrk and cyc01 mutanata koncentracija H2O2 raste nakon svake doze vMETH-a. To ukazuje da bez obzira na mutaciju, METH uzrokuje porast oksidativnih vrsta i postavlja pitanja o molekularnim mehanizmima uključenim u regulaciju redoks stanja nakon izlaganja psihostimulansima