Accumulation of diacylglycerol induced by CCl_4-derived radicals in rat liver membrane and its inhibition with radical trapping reagent : FT-IR spectroscopic and HPLC chromatographic observations

Abstract

We have investigated the accumulation of diacylglycerol (DAG) induced by carbon tetrachloride (CCl_4)-derived radicals in the liver of female Sprague-Dawley (SD) rats after intraperitoneally injecting CCl_4. DAG is an intracellular activator of protein kinase C (PKC) which regulates cell proliferation and differentiation. The electron spin resonance (ESR) study gave the signal of the PBN-CCl_3 adduct in the liver of the rats which were pretreated with PBN, confirming that CCl_4 was metabolized into CCl_3 radicals with cytochrome P450 enzyme and indicating that PBN could trap them. The blood biochemical assay supported the trapping of the CCl_3 radicals ; the pretreatment of rats with PBN inhibited the increase in the GOT and GPT values upon exposure to CCl_4. The Fourier transform-infrared (FT-IR) study indicated in comparison with the model compounds that the CCl_4-injected rats accumulated DAG in addition to phosphatidylcholine, phosphatidylethanolamine and triglyceride (TG) in the lipid membrane fraction of the liver homogenate. DAG was found to be ca. 10-15 % of the membrane phospholipids by weight. However, DAG was not found in the lipid of the liver microsomes, suggesting that it is formed only in the cell membrane of liver. Also, neither DAG nor TG was found in the lipid membrane of the rats that were pretreated with PBN followed by an injection of CCl_4. The formation of DAG was confirmed by an HPLC study. The activation of PKC was observed in liver homogenate in the rats that were injected with CCl_4. On the basis of the above findings, it was concluded that the CCl_4-derived radicals stimulate PKC through the accumulation of DAG in the liver membrane of the rats. Furthermore, it was shown that PBN has a protective and therapeutic effect against CCl_4-induced damage