Doxorubicin induces ceramide and diacylglycerol accumulation in rat hepatocytes through independent routes.

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its toxicity. This toxicity has been associated with free radicals generated during the drug metabolism. We previously found that DOX increased the intracellular diacylglycerol (DAG) levels at 1h in isolated rat hepatocytes, probably by mobilizing choline-enriched phospholipids. In this work, we studied the effects of DOX on oxidative stress markers, and the possible contribution of ceramide metabolism to DAG accumulation. Other possible routes of DAG production, such as impairment of triacylglycerol (TAG) synthesis, and their connection with oxidative stress were also investigated. Time-course experiments revealed that DOX decreased intracellular GSH at 2h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [(3)H]-ceramide during the first 90 min of exposure, but increased it significantly at 2h. [(3)H]-Sphingomyelin remained unchanged during the whole period. These results indicate that ceramide metabolism is not involved in the early DAG response to DOX. The drug markedly increased the incorporation of [(3)H]-oleate into intracellular DAG from 60 min. In contrast, DOX reduced the incorporation of [(3)H]-oleate into intracellular phospholipids and TAG. DOX inhibited TAG synthesis at the DAG acyltransferase step. These results suggest that DOX increases the intracellular levels of the lipid messengers, ceramide and DAG, by independent mechanisms. Activation of the de novo synthesis of ceramide is probably involved in the sphingolipid accumulation, while inhibition of TAG synthesis contributes to DAG accumulation, this response being independent of oxidative damage.

Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: involvement of lipid peroxidation.

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [(3)H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with alpha-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress.

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.