R. Carini

Lipid peroxidation and irreversible damage in the rat hepatocyte model: Protection by the silybin-phospholipid complex IdB 1016

IdB 1016 is a new silybin-phospholipid complex which is more bioavailable than the flavonoid silybin itself and displays free radical scavenging and antioxidant properties in liver microsomes. We report here that the addition of increasing concentrations of IdB 1016 to isolated rat hepatocytes caused a dose-dependent inhibition of lipid peroxidation induced by ADP-Fe3+ or cumene hydroperoxide. Moreover, IdB 1016 at the concentration which completely prevented MDA formation also protected isolated hepatocytes against the toxicity of pro-oxidant agents such as allyl alcohol, cumene hydroperoxide and bromotrichloromethane, without interfering with the activation mechanism of these xenobiotics. Similar protection was also obtained in hepatocytes prepared from animals pretreated in vivo with IdB 1016 while rat supplementation with pure silybin was totally inefficient. These results indicate IdB 1016 as being a potentially useful protective agent against free radical-mediated toxic liver injury.

Comparative evaluation of the antioxidant activity of α-tocopherol, α-tocopherol polyethylene glycol 1000 succinate and α-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions

Abstract The antioxidant activity of α-tocopherol polyethylene glycol 1000 succinate (TPGS) and of α-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from rat liver. Both TPGS and TS require esterase activity to yield free α-tocopherol and, hence, antioxidant activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent amount of directly-added free α-tocopherol. The low antioxidant efficiency of directly added free α-tocopherol in such water-based experimental systems as used here seems to be due to its extreme hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined as a useful source of α-tocopherol in certain clinical situations and is here shown to be a convenient and effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.

Stimulation of lipid peroxidation increases the intracellular calcium content of isolated hepatocytes

Lipid peroxidation induced in isolated rat hepatocytes by FeCl3 (0.1 mM) was associated with an increase in the cytosolic free Ca2+ and in the ionophore-mobilizable Ca2+ content of both mitochondrial and extramitochondrial (endoplasmic reticular) pools. Ca2+ accumulation was completely prevented by the antioxidants promethazine and vitamin E succinate and was not linked to the inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport or to the depletion of intracellular ATP content. Moreover, preincubation of the hepatocytes with the Ca2+ channel blockers verapamil and nifedipine inhibited the elevation of cytosolic Ca2+, as well as the ion accumulation without interfering with the stimulation of lipid peroxidation by iron. These results suggest that peroxidative alterations of the hepatocyte plasma membranes might perturb the functions of verapamil- and nifedipine-sensitive Ca2+ channels resulting in a net influx of Ca2+, which is subsequently sequestrated in the intracellular compartments.

Studies on the antioxidant and free radical scavenging properties of IdB 1016 a new flavanolignan complex.

Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.

Mechanisms responsible for carbon tetrachloride-induced perturbation of mitochondrial calcium homeostasis

Incubation of isolated hepatocytes with CCl4 results in early reduction of the intracellular calcium content, mostly due to loss from the mitochondrial compartment. CCl4 treatment directly affects mitochondrial functions as indicated by the inhibition of Ca2+ uptake in cells permeabilized to the ion by digitonin exposure and by the reduction of intracellular ATP content in hepatocytes incubated in a glucose‐free medium. Such mitochondrial damage is not caused by CCl4‐induced stimulation of lipid peroxidation since it is not prevented by α‐tocopherol, used at a concentration able to inhibit completely peroxidative reactions without interfering with CCl4 activation. All data together are in favour of a direct action of CCl4‐reactive metabolites on liver cell calcium homeostasis.

Effects of carbon tetrachloride on calcium homeostasis: A critical reconsideration

The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.

Ethanol potentiates hypoxic liver injury: role of hepatocyte Na+ overload

Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na(+) homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na(+) levels preceded cell killing and Na(+) levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na(+) increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO(3)(-)-free buffer or in the presence of Na(+)/H(+) exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na(+) influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na(+) accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na(+) influx caused cytotoxicity in hepatocytes pre-treated with Na(+), K(+)-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na(+) accumulation.

Pro-Hemolytic Effect of Aldehydic Products of Lipid Peroxidation

In order to evaluate the pro-hemolytic action exerted by different classes of biogenic aldehydes, normal red cells obtained from human beings of both sexes were incubated at 37 degrees C under iso or hypo-osmotic conditions in the presence of hydroxyalkenals or alkanals, in a concentration compatible with those actually recovered during red cell lipid peroxidation. None of the tested aldehydes showed a direct hemolytic effect, i.e. red cell lysis in iso-osmotic conditions. Conversely, almost all assayed alkanals and hydroxyalkenals exhibited a pre-lytic damage of human erythrocytes, as detected in the red cells suspended in hypo-osmotic medium. The highest pro-hemolytic effect was displayed by hexanal, nonanal, 2-nonenal and 4-hydroxynonenal.

Alterations of hepatocyte Ca2+ homeostasis by triethylated lead (Et3Pb+): are they correlated with cytotoxicity?

Isolated rat hepatocytes were used to investigate the biochemical mechanisms of toxicity of triethyllead (Et3Pb+), a highly neurotoxic degradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 microM Et3Pb+ to hepatocyte suspensions a decrease of mitochondrial membrane potential and of the capacity of mitochondria and microsomes to retain Ca2+ occurred. A dose-dependent release of mitochondrial Ca2+ as well as an inhibition of microsomal Ca(2+)-ATPase activity were also evident when Et3Pb+ (from 2.5 microM up to 50 microM) was added to, respectively, isolated liver mitochondria and microsomes. Further experiments using hepatocytes loaded with the Ca2+ indicator Fura-2AM demonstrate that 1 min from addition of Et3Pb+ the cytosolic free Ca2+ levels increased by about 3-fold. High affinity plasma membrane Ca(2+)-ATPase activity was also significantly inhibited in hepatocytes treated with Et3Pb+, suggesting that an impairement of the mechanisms controlling the efflux of extracellular Ca2+ was concomitantly involved in the rise in cytosolic Ca2+ concentration. The increase in the cytosolic Ca2+ levels caused by Et3Pb+ was followed by a rapid decline of cell viability. However, the addition of EGTA or of the intracellular Ca2+ chelator BAPTA/AM did not affect either the time-course or the extent of cytotoxicity. Conversely, fructose, a glycolytic substrate that was able to support ATP production, prevented hepatocyte death. Thus, the depletion of cellular energy stores rather than the increase in cytosolic Ca2+ appears to be the mechanism by which Et3Pb+ causes irreversible injury in isolated hepatocytes.

ACTIVATION OF HUMAN IMMUNODEFICIENCY VIRUS LONG TERMINAL REPEAT BY ARACHIDONIC ACID

Arachidonic acid is the precursor of highly reactive mediators, including prostaglandins and leukotrienes, and the most abundant n-6 polyunsaturated fatty acid in mammalian cell membranes. It is released from phospholipids upon many inflammatory stimuli. In this study, a chloramphenicol acyltransferase reporter gene, under control of the human immunodeficiency virus-1 long terminal repeat, was strongly induced upon treating human promonocytes with arachidonic acid. The n-3 fatty acid eicosapentenoic, found in abundance in fish oil, had no effect. HIV-1 long terminal repeat activation by arachidonic acid was suppressed by inhibitors of both lipoxygenase and cyclooxygenase pathways, suggesting that metabolites, rather than arachidonic acid itself, mediated the stimulatory effect. This is the first report linking HIV-1 expression to the metabolism of arachidonic acid.