Mechanism of free radical generation in platelets and primary hepatocytes: A novel electron spin resonance study

Abstract

Oxygen free radicals have been implicated in the pathogenesis of toxic liver injury and are thought to be involved in cardiac dysfunction in the cirrhotic heart. Therefore, direct evidence for the electron spin resonance (ESR) detection of how D‑galactosamine (GalN), an established experimental hepatotoxic substance, induced free radicals formation in platelets and primary hepatocytes is presented in the present study. ESR results demonstrated that GalN induced hydroxyl radicals (OH•) in a resting human platelet suspension; however, radicals were not produced in a cell free Fenton reaction system. The GalN‑induced OH• formation was significantly inhibited by the cyclooxygenase (COX) inhibitor indomethasin, though it was not affected by the lipoxygenase (LOX) or cytochrome P450 inhibitors, AA861 and 1‑aminobenzotriazole (ABT), in platelets. In addition, the present study demonstrated that baicalein induced semiquinone free radicals in platelets, which were significantly reduced by the COX inhibitor without affecting the formed OH•. In the mouse primary hepatocytes, the formation of arachidonic acid (AA) induced carbon‑centered radicals that were concentration dependently enhanced by GalN. These radicals were inhibited by AA861, though not affected by indomethasin or ABT. In addition, GalN did not induce platelet aggregation prior to or following collagen pretreatment in human platelets. The results of the present study indicated that GalN and baicalein may induce OH• by COX and LOX in human platelets. GalN also potentiated AA induced carbon‑centered radicals in hepatocytes via cytochrome P450. The present study presented the role of free radicals in the pathophysiological association between platelets and hepatocytes.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2–PKC–AKT Activation in Human Platelets

“…In addition, hydrogen peroxide formation in platelets is converted into hydroxyl radicals. During the initial phase of platelet activation, free radicals act as secondary signals, and PKC is involved in receptor-mediated free radical production in platelets [36]. Esculetin possesses a structural coumarin moiety and two hydroxyl substitutions, and it has potent antioxidant activity; evidence has been presented for its ability to quench free radical such as DPPH radical and hydroxyl radicals [9].…”