hrvatski

Učinak flavonoida na toksično djelovanje okratoksina A

Okratoksin A (OTA) je široko rasprostranjen mikotoksin kojeg proizvode pojedine vrste plijesni roda Aspergillus i Penicillium. U serumu ljudi na područjima gdje se javlja endemska nefropatija (EN) ustanovljene su relativno velike koncentracije OTA. Budući da pojavnost visokih vrijednosti OTA u serumu nije ograničena samo na ta područja, preostaje istražiti svezu između izlaganja ovom mikotoksinu i EN. Pokusi na životinjama pokazuju kako je OTA snažni nefrotoksični i kancerogeni agens. Nadalje, nepovoljno utječe na zgrušavanje krvi i na imunološki odgovor. Oksidatvni stres kojeg uzrokuje OTA ukazuje na njegov genotoksični učinak te učinak na apoptozu, koji očito imaju značajnu ulogu u razvoju kronične tubulointersticijske nefropatije. S druge strane, flavonoidi su poznati po svojim antiinflamatornim, antitumorskim, kardioprotektivnim i imunomodulacijskim aktivnostima. Smatra se da su neki od navedenih bioloških učinaka povezani s njihovim antioksidativnim svojstvima koja se zasnivaju na ograničavanju stvaranja reaktivnih kisikovih spojeva i/ili njihovom vezivanju. Cilj ovog rada bio je ispitati djelovanje flavonoida na stanične obrambene mehanizme u stanicama tretiranih s OTA. MDCK, LLC-PK1, HeLa i K562 stanične linije izložene su kvercetinu, kemferolu, miricetinu, naringeninu i apigeninu u koncentracijama 1-10 mikroM i/ili različitim dozama OTA (0, 1-25 mikroM) u hranjivom mediju tijekom 24 sata. Ispitivali smo učinke OTA i flavonoida na viabilnost stanica i na vrste stanične smrti (apoptoza ili nekroza), ukupni antioksidativni status, na razinu reduciranog-oksidiranog GSH-GSSG te na koncentraciju staničnog ATP-a. Također je DPPH• metodom određena antiradikalna aktivnost flavonoida s različitim strukturnim obilježjima. Sniženi ukupni antioksidativni status tijekom izlaganja OTA naglašava ulogu staničnih antioksidansa u mehanizmima stanične obrane prema oksidacijskom stresu. Svi ispitivani flavonoidi imali su sposobnost “ hvatanja“ DPPH• radikala, ali su pokazali učinke različitih intenziteta. Najveću antiradikalnu aktivnost pokazao je miricetin, a zatim kvercetin. Kod MDCK stanica tretiranih s OTA miricetin je bio najučinkovitiji u zaštiti živih stanica. Kemferol i miricetin su više od ostalih flavonoida djelovali pozitivno na vijabilnost K562 stanica. Zaključili smo da se primjena antioksidativnih adjuvansa poput flavonoida može primjeniti kao preventivno djelovanje ili kao terapijska intervencija kod toksičnog djelovanja OTA. Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of fungi Aspergillus and Penicillium. OTA was found in relative high concentrations in human sera in areas where Endemic Nephropathy (EN) occurs. Since the occurrence of high serum OTA is not restricted to EN areas the link between OTA exposure and EN remains yet to be verified. However, animal experiments show that OTA is a potent nephrotoxic and carcinogenic agent. In addition, it adversely affects blood coagulation and the immune response. OTA-induced oxidative stress may explain its genotoxic effects and apoptosis, which in turn are likely to play important roles in the development of chronic tubulointerstitial nephritis. On the other hand, many flavonoids are known for their anti-inflamatory, antitumoral, antialergic, cardioprotective and immunomodulatory activities. Several of these biological activities are assumed to be connected with antioxidant properties of these compounds, which are displayed by limiting the production of reactive oxigen species and/or scavenging them. The aim of this study was to investigate the cell defence impact of flavonoids in OTA treated cell lines. MDCK, LLC-PK1, HeLa and K562 cell lines were exposed to 1-10 microM of quercetin, kaempferol, myricetin naringenin and apigenin and /or the different doses of OTA (0.1 to 25 microM) in culture medium for 24 hrs. We studied the effects of OTA and flavonoids on cell viability and the type of cell death (apoptosis or necrosis), total antioxidative status, reduced-oxidized GSH-GSSG levels and intracellular ATP concentration. The free radical scavenging ability of flavonoids with different substitution patterns were also tested. We used DPPH• radical scavenging assay. The decreased total antioxidant status during exposure to OTA would emphasise the role of cellular antioxidants in the cell defence mechanisms against oxidative stress. All investigated flavonoids possessed scavenging activity against DPPH• radicals, but they exhibited effects of different magnitude. The maximal antiradical activity was recorded for myricetin and quercetin, respectively. In OTA treated MDCK cells myricetin was the most efficient in defence of living cells. Kaempferol and myricetin positively affected cell viability of K562 cells, more than other flavonoids. We conclude that the administration of antioxidative adjuvants such as flavonoids could be used as a preventive action or as a therapeutic intervention in OTA-induced toxicity.

oksidativni stres; antioksidansi; flavonoidi; stanični GSH-GSSG; stanični ATP; apoptoza; okratoksin A

nije evidentirano

engleski

Effects of flavonoids in ochratoxin A-induced toxicity

Okratoksin A (OTA) je široko rasprostranjen mikotoksin kojeg proizvode pojedine vrste plijesni roda Aspergillus i Penicillium. U serumu ljudi na područjima gdje se javlja endemska nefropatija (EN) ustanovljene su relativno velike koncentracije OTA. Budući da pojavnost visokih vrijednosti OTA u serumu nije ograničena samo na ta područja, preostaje istražiti svezu između izlaganja ovom mikotoksinu i EN. Pokusi na životinjama pokazuju kako je OTA snažni nefrotoksični i kancerogeni agens. Nadalje, nepovoljno utječe na zgrušavanje krvi i na imunološki odgovor. Oksidatvni stres kojeg uzrokuje OTA ukazuje na njegov genotoksični učinak te učinak na apoptozu, koji očito imaju značajnu ulogu u razvoju kronične tubulointersticijske nefropatije. S druge strane, flavonoidi su poznati po svojim antiinflamatornim, antitumorskim, kardioprotektivnim i imunomodulacijskim aktivnostima. Smatra se da su neki od navedenih bioloških učinaka povezani s njihovim antioksidativnim svojstvima koja se zasnivaju na ograničavanju stvaranja reaktivnih kisikovih spojeva i/ili njihovom vezivanju. Cilj ovog rada bio je ispitati djelovanje flavonoida na stanične obrambene mehanizme u stanicama tretiranih s OTA. MDCK, LLC-PK1, HeLa i K562 stanične linije izložene su kvercetinu, kemferolu, miricetinu, naringeninu i apigeninu u koncentracijama 1-10 mikroM i/ili različitim dozama OTA (0, 1-25 mikroM) u hranjivom mediju tijekom 24 sata. Ispitivali smo učinke OTA i flavonoida na viabilnost stanica i na vrste stanične smrti (apoptoza ili nekroza), ukupni antioksidativni status, na razinu reduciranog-oksidiranog GSH-GSSG te na koncentraciju staničnog ATP-a. Također je DPPH• metodom određena antiradikalna aktivnost flavonoida s različitim strukturnim obilježjima. Sniženi ukupni antioksidativni status tijekom izlaganja OTA naglašava ulogu staničnih antioksidansa u mehanizmima stanične obrane prema oksidacijskom stresu. Svi ispitivani flavonoidi imali su sposobnost “ hvatanja“ DPPH• radikala, ali su pokazali učinke različitih intenziteta. Najveću antiradikalnu aktivnost pokazao je miricetin, a zatim kvercetin. Kod MDCK stanica tretiranih s OTA miricetin je bio najučinkovitiji u zaštiti živih stanica. Kemferol i miricetin su više od ostalih flavonoida djelovali pozitivno na vijabilnost K562 stanica. Zaključili smo da se primjena antioksidativnih adjuvansa poput flavonoida može primjeniti kao preventivno djelovanje ili kao terapijska intervencija kod toksičnog djelovanja OTA. Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of fungi Aspergillus and Penicillium. OTA was found in relative high concentrations in human sera in areas where Endemic Nephropathy (EN) occurs. Since the occurrence of high serum OTA is not restricted to EN areas the link between OTA exposure and EN remains yet to be verified. However, animal experiments show that OTA is a potent nephrotoxic and carcinogenic agent. In addition, it adversely affects blood coagulation and the immune response. OTA-induced oxidative stress may explain its genotoxic effects and apoptosis, which in turn are likely to play important roles in the development of chronic tubulointerstitial nephritis. On the other hand, many flavonoids are known for their anti-inflamatory, antitumoral, antialergic, cardioprotective and immunomodulatory activities. Several of these biological activities are assumed to be connected with antioxidant properties of these compounds, which are displayed by limiting the production of reactive oxigen species and/or scavenging them. The aim of this study was to investigate the cell defence impact of flavonoids in OTA treated cell lines. MDCK, LLC-PK1, HeLa and K562 cell lines were exposed to 1-10 microM of quercetin, kaempferol, myricetin naringenin and apigenin and /or the different doses of OTA (0.1 to 25 microM) in culture medium for 24 hrs. We studied the effects of OTA and flavonoids on cell viability and the type of cell death (apoptosis or necrosis), total antioxidative status, reduced-oxidized GSH-GSSG levels and intracellular ATP concentration. The free radical scavenging ability of flavonoids with different substitution patterns were also tested. We used DPPH• radical scavenging assay. The decreased total antioxidant status during exposure to OTA would emphasise the role of cellular antioxidants in the cell defence mechanisms against oxidative stress. All investigated flavonoids possessed scavenging activity against DPPH• radicals, but they exhibited effects of different magnitude. The maximal antiradical activity was recorded for myricetin and quercetin, respectively. In OTA treated MDCK cells myricetin was the most efficient in defence of living cells. Kaempferol and myricetin positively affected cell viability of K562 cells, more than other flavonoids. We conclude that the administration of antioxidative adjuvants such as flavonoids could be used as a preventive action or as a therapeutic intervention in OTA-induced toxicity.

oxidative stress; antioxidants; flavonoids; intracellular GSH-GSSG; intracellular ATP; apoptosis; ochratoxin A

nije evidentirano