| Abstract | 2-metoksi-1,4-naftokinon (2-MNQ) prirodni je spoj za kojega je pokazano antifungalno, antibakterijsko i antitumorno
djelovanje. Stoga je cilj ovog rada bio ispitati toksičnost i oksidacijski stres kao mogući mehanizam toksičnosti 2-MNQ na biljnom
modelu. Kao modelni organizam odabrana je bijela gorušica (Sinapis alba L.).
Sjemenke bijele gorušice bile su izložene 2-MNQ u koncentracijama 1, 5, 10, 20, 30 i 40 μg/mL kroz 3 dana. U pokus su
bile uključene negativna kontrola (sjemenke bijele gorušice izložene 3 dana destiliranoj vodi) i pozitivna kontrola (sjemenke bijele gorušice izložene 3 dana 0,02 M otopini CuSO4). Nakon 3-dnevne izloženosti izvagana je masa svježega tkiva te su pripremljeni homogenati klijanaca kako bi se u njima odredila razina parametra oksidacijskog stresa, glutationa (GSH) i malondialdehida (MDA). Razina GSH određena je pomoću DTNB reagensa spektrofotometrijski pri valnoj duljini od 412 nm. MDA je kvantificiran u reakciji s 2-tiobarbituratnom kiselinom, a razina MDA određena je spektrofotometrijski pri 532 nm. Dobiveni rezultati statistički su obrađeni t-testom, a razina značajnosti postavljena je na P ≤ 0,05. 3-dnevna izloženost klijanaca bijele gorušice 2-MNQ dovela je do smanjenja mase svježega tkiva. 2-MNQ (10 μg/mL) značajno je smanjio masu svježega tkiva u odnosu na negativnu kontrolu (287 ± 11,53 mg vs. 343,67 ± 39,55 mg; P < 0,05). S porastom koncentracije 2-MNQ zabilježen je daljnji pad mase svježega tkiva. 3-dnevna izloženost 2-MNQ u koncentraciji 1 μg/mL značajno je smanjila razinu GSH u odnosu na negativnu kontrolu (2,92 ± 0,48 μM vs. 4,55 ± 0,67 μM; P < 0,05). Više koncentracije 2-MNQ značajno su povisile razinu GSH. Za pretpostaviti je da su klijanci bijele gorušice povećali sintezu GSH kako bi se zaštitili od djelovanja 2-MNQ. S druge strane, 3-dnevna izloženost klijanaca 2-MNQ dovela je do pada razine MDA. U klijanaca tretiranih s 2-MNQ (10 μg/mL) razina MDA je bila 0,07 ± 0,02 μM što je bilo značajno manje od negativne kontrole (0,11 ± 0,03 μM; P < 0,05). Iako je bilo za očekivati da će zbog stresa doći do porasta razine MDA, pad razine MDA je moguće objasniti pojačanom sintezom GSH koja je spriječila pojavu lipidne peroksidacije. S obzirom da je došlo do smanjenja mase svježega tkiva klijanaca bijele gorušice nakon izloženosti 2-MNQ može se zaključiti da je 2-MNQ fitotoksičan. Izloženost 2-MNQ dovela je do promijene u razini parametara oksidacijskog stresa te se može zaključiti da je makar djelomično u fitotoksičan učinak 2-MNQ uključen oksidacijski stres. Također, ovi rezultati pokazuju da je biljni model prihvatljiv u pretklinički istraživanjima zbog etičkih i ekonomskih razloga. |
| Abstract (english) | 2-methoxy-1,4-naphthoquinone (2-MNQ) is a natural compound for which antifungal, antibacterial and antitumor activity
has been shown. Therefore, the aim of this study was to examine toxicity and oxidative stress as a possible mechanism of 2-MNQ toxicity in a plant model. White mustard (Sinapis alba L.) was selected as the model organism.
White mustard seeds were exposed to 2-MNQ at concentrations of 1, 5, 10, 20, 30 and 40 μg/mL for 3 days. A negative
control (white mustard seeds exposed to distilled water for 3 days) and a positive control (white mustard seeds exposed to 0.02 M CuSO4 solution for 3 days) were included in the experiment. After 3 days of exposure, fresh tissue weight was determined and seedling homogenates were prepared in order to assess the level of oxidative stress parameters, glutathione (GSH) and malondialdehyde (MDA). GSH level was determined using DTNB reagent spectrophotometrically at a wavelength of 412 nm. MDA was quantified by reaction with 2-thiobarbiturate acid, and the MDA level was determined spectrophotometrically at 532 nm. For statistical analysis ttest was used and the significance level was set at P ≤ 0.05.
After 3-day exposure of white mustard seedlings to 2-MNQ a reduction in fresh tissue weight was recorded. 2-MNQ (10
μg/mL) significantly reduced fresh tissue weight compared to the negative control (287 ± 11.53 mg vs. 343.67 ± 39.55 mg; P <0.05). With the increase of 2-MNQ concentration, a further decrease in fresh tissue weight was observed. 3-day exposure to 2-MNQ at a concentration of 1 μg/mL significantly reduced GSH level in comparison to the negative control (2.92 ± 0.48 μM vs. 4.55 ± 0.67 μM; P <0.05). Higher 2-MNQ concentrations significantly increased GSH levels. It can be assumed that white mustard seedlings increased GSH synthesis in order to protect themselves against the action of 2-MNQ. On the other hand, 3-day exposure of seedlings to 2-MNQ led to a decrease in MDA level. In seedlings treated with 2-MNQ (10 μg/mL) the MDA level was 0.07 ± 0.02 μM which was significantly lower than the MDA level in the negative control (0.11 ± 0.03 μM; P <0.05). Although due to the stress an increase in MDA level was expected, a decrease occurred. The decrease in MDA level can be explained by the increased synthesis of GSH in plant tissue which prevented the occurrence of lipid peroxidation. Since exposure of white mustard seedlings to 2-MNQ led to a decrease of fresh tissue weight, it can be concluded that 2- MNQ is phytotoxic. Additionally, exposure to 2-MNQ led to a change in the level of oxidative stress parameters, thus oxidative stress is at least partially involved in the phytotoxic effect of 2-MNQ. Also, the results of this study show that the plant model is acceptable in preclinical studies due to ethical and economic reasons. |
