Microsomal sphingomyelin accumulation in thioacetamide-injured regenerating rat liver: involvement of sphingomyelin synthase activity.

The purpose of this work was to determine whether alterations in the lipid composition of rat liver microsomal membranes existed during thioacetamide-induced injury prior to the development of hepatic cancer and biochemical mechanisms involved. Rats were injected intraperitoneally with (50 mg/kg body wt per day) thioacetamide or diluent for 8 days. Liver homogenates and microsomal membranes from liver homogenates were obtained. Incorporation of [32P]orthophosphate into whole liver lipids and hepatic microsomal lipids was evaluated 75 min after isotope administration. These determinations were made after two separate periods of treatment (3 and 8 days). Activity of sphingomyelin synthase was assayed in rat liver homogenates as well as in the purified microsomal fractions. Results demonstrated a maintenance of liver and hepatic microsomal contents of phosphatidylcholine during thioacetamide-induced injury even when the biosynthesis of this glycerophospholipid in both liver and their microsomal fractions appeared decreased. Also observed was a considerable increase of microsomal sphingomyelin, as well as an increased hepatic biosynthesis of sphingomyelin caused by thioacetamide treatment. The microsomal sphingomyelin/phosphatidylcholine radioactivity ratio significantly increased. Sphingomyelin synthase activity in liver homogenate appeared stimulated. In conclusion, our data are consistent with a thioacetamide-induced increase in microsomal sphingomyelin by a stimulation of sphingomyelin synthase. Based on this and previous studies, accumulation of sphingomyelin in the microsomal purified fraction is associated with the number of thioacetamide doses and is an early event clearly detected prior to tumoral characteristics of hepatocytes.

Thioacetamide alters the regulation of cytidylyltransferase activity.

Using the weak hepatocarcinogen thioacetamide, we found a 73% decrease in microsomal CTP: Phosphocholine cytidylyltransferase (CT) activity after two months of treatment. We investigated the effect of different effectors on this enzyme activity. Results show that incubation of liver homogenates of treated animals in the presence of either oleate or spermine restored control values of microsomal activity. Incubation of thioacetamide homogenates in the presence of cAMP showed no changes in CT activity, while incubation of control and thioacetamide homogenates in the presence of Ca2+ induced an activation in microsomal activity, although of less intensity in thioacetamide homogenates than in control preparations. Results suggest that thioacetamide alters the regulation of cytidylyltransferase activity.

Alterations in lipid characteristics of lysosomes are involved in liver necrosis induced by thioacetamide.

To determine whether lysosomal lipid composition is altered in hepatic necrosis, we studied this parameter in thioacetamide-induced necrosis and in its regenerating stage as well as in the recovery of thioacetamide-induced injury. Results showed that in liver necrosis there is an increase in the protein and phospholipid lysosomal contents. This effect may be related to an increased number of lysosomes. These organelles also suffered a decrease in cholesterol content. When liver necrosis was recovered either pharmacologically or spontaneously all three parameters recovered their normal values. These results suggest that lysosomes and their lipid composition play a role in progression of hepatic necrosis.

Hepatic expression of apolipoprotein A-I gene in rats is upregulated by monounsaturated fatty acid diet.

The effect of the degree of dietary fat saturation on the hepatic expression of apolipoprotein A-I mRNA was studied in male rats. Animals were maintained for two months on a high fat diet (40% w/w) containing 0.1% cholesterol. Two groups of control animals received either chow diet or chow plus 0.1% cholesterol, while experimental groups received their fat supplement as coconut, corn or olive oil respectively. Dietary cholesterol did not affect apolipoprotein A-I mRNA levels as compared to control animals. Corn oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA than those receiving cholesterol, or coconut oil plus cholesterol. Olive oil fed animals had significantly higher levels of hepatic apolipoprotein A-I mRNA when compared to all other dietary groups. Our data indicate that monounsaturated fatty acids supplied as olive oil play a major role in regulating the hepatic expression of apolipoprotein A-I in male rats.

Effect of acute thioacetamide administration on rat brain phospholipid metabolism.

Brain phospholipid composition and the [32P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.

Association between rat serum cholinesterase and some phospholipid components of lipoproteins in thioacetamide-induced hepatic injury.

Rat serum lipoprotein phospholipids and serum cholinesterase activity in control and thioacetamide-treated rats (50 mg/kg/day for 30 days) were studied. Analyses were done after 1, 3, 8 and 30 intraperitoneal doses of thioacetamide or 0.15 mol/l NaCl. Cholinesterase activity significantly increased with thioacetamide treatment. Only two phospholipids: LDL-phosphatidylcholine and HDL-lysophosphatidylcholine appeared associated with cholinesterase activity. LDL-phosphatidylcholine increased through the action of hepatotoxic thioacetamide while HDL-lysophosphatidylcholine significantly decreased. Because of the high statistically significant association between changes in these lipoprotein phospholipids and in cholinesterase in this model of hepatic injury, we conclude that cholinesterase could be involved in the regulation of these phospholipid levels.

Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol.

Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% (w/w) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and beta-D-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.

Inhibition of the translocation of cytidylyltransferase can be a delayed mechanism to control phosphatidylcholine biosynthesis "in vivo".

To determine whether CTP: Phosphocholine cytidylyltransferase is responsible for the previously observed decrease in phosphatidylcholine biosynthesis during thioacetamide treatment, rats were injected daily i.p. with either this agent (50 mg/kg/day) or diluent for 3 and 60 days. Microsomal and cytosolic enzymatic activities were estimated. Results demonstrated that cytosolic enzyme activity appears significantly decreased; however microsomal enzyme only appears significantly decreased after 60 days of treatment. At this time period microsomal percentage of enzyme activity is also significantly decreased. From these results it can be deduced that inhibition of enzyme translocation can be a delayed adaptative response "in vivo".

Changes in serum cholinesterase (EC 3.1.1.8) activity in rats consuming a high-fat diet.

Adult male rats were fed on a control diet containing (g/kg) carbohydrate 600, lipid 35 and protein 190, or on a high-fat diet containing carbohydrate 360, lipid 420 and protein 120. After 30 d, the high-fat diet provoked a decrease in serum cholinesterase (EC 3.1.1.8) activity which was reversed by feeding rats on the control diet. The observed decrease after 90 d on the high-fat diet was not seen if a simultaneous daily intraperitoneal injection of a lipotrophic agent containing (mg/kg) S-adenosyl-L-methionine 3, coenzyme A 0.1, UDP-glucose 30 and CDP-choline 1.5 was given to rats on the high-fat diet. The findings are discussed in relation to the apparent susceptibility of serum cholinesterase to dietary components and its possible role in lipid metabolism.

Lyso-phosphatidylcholine is implicated in thioacetamide-induced liver necrosis.

Thioacetamide is a weak hepatocarcinogen. To determine whether alterations in lysophosphatidylcholine are implicated in thioacetamide-induced hepatic necrosis, rats were injected i.p. with this agent (50 mg/Kg body weight per day) or diluent for 1, 3, 8 and 30 days. Serum catalytic activities of aminotransferases were determined. Incorporation of (32P)-orthophosphate into hepatic lysophosphatidylcholine was also evaluated in animals killed 75 minutes or 13 hours after isotope administration. Results demonstrate that: A significant increase in hepatic lysolecithin concentration occurs when a maximum level of serum aminotransferases is present. An increase of (32P)-orthophosphate radioactive incorporation in lysolecithin was observed at the two assayed labelling periods, which suggest an activation of phospholipase A. The radioactivity present in lysolecithin after 13 h isotope injection showed a close correlation with serum level of aminotransferases. From these results it can be deduced that lysolecithin is implicated in TAA-induced necrosis and may be generated by increase in either phospholipase A activity and/or synthesis.

Effect of S-adenosyl-L-methionine on thioacetamide-induced liver damage in rats.

S-Adenosyl-L-methionine (Ado-met) administration to rats significantly improved liver necrosis induced by thioacetamide (TAA) as evidenced by reduction of TAA-elevated catalytic activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALAT). Ado-met, however, was not effective in reduction of catalytic activity of serum alkaline phosphatase (ALP) which increased as a consequence of TAA administration. Histologic analysis of the livers supported the biochemical data. Hepatocellular damage was evident from the first day of TAA treatment at daily (i.p.) doses of 50 mg/kg body wt. Maximal necrosis was apparent after 3 days of TAA administration. When rats were treated once a day, for 3 days with Ado-met (2 mg/kg body wt) as well as with TAA, significant reduction of hepatic necrotic area was observed. A similar effect was obtained when doses of 200 mg/kg body wt. of Ado-met were utilized.

[Urea and arginase activity in rat liver during the administration of thioacetamide]. / Urea y actividad argintasica en hígado de rata durante la administración de tioacetamida (TAM).

The authors measure the concentration of urea and arginase activity at pH 6.5 and 9.7 in the rat liver at intervals during the administration thioacetamide (TAM) at a dose of 50 mg per kg of body weight and per day. The values obtained correlate the amount of urea and the arginase activity at pH 6.5 and 9.7 during TAM administration for the intervals comprised between 1-12 days and 12-30 days. These results suggest that the concentration of liver urea depends mainly of the level at arginase activity, which is regulated by the proton concentration of the internal environment.

[Effect of pH and activation on the arginase activity of rat liver treated with thioacetamide]. / Efecto del pH y activation sobre la actividad arginásica de hígado de rata tratada con tioacetamida.

Arginase (E.C. 3.5.3.1) activity of liver from thioacetamide treated rats has been investigated in relation to 6.5 pH in the reaction medium, as well as stability against activation process. The results suggest that thioacetamide damage affects enzyme protein at the level of ionizing groups of active center and/or binding to Mn2+.

[Effect of thioacetamide on the incorporation of 32P into phospholipids of rat liver]. / Efecto de la tioacetamida sobre la incorporación de 32P a fosfolípidos de hígado de rata.

The effect of thioacetamide on rat liver phospholipids biosynthesis was investigated using 32P as precursor. The incorporation of 32P-ortophosphate in the polar lipid fraction and the specific radioactivities of individual phospholipids in the liver fraction and the specific radioactivities of individual phospholipids in the liver of control rats and rats treated with thioacetamide were determined 75 min after intraperitoneal administration of 32P-ortophosphate. Intraperitoneal injection of thioacetamide (daily dose of 100 mg/kg body weight) in male Wistar rats resulted in an increase of 32P incorporation in the over-all phospholipids, after 8 doses administration and a significant decrease during the chronic intoxication. Specific activity of phosphatidylcholine decreased and a parellel increase in specific radioactivity of lisophosphatidylcholine was found after three days of thioacetamide treatment. There was also observed a marked increase in specific radioactivity of sphingomyelin, which could be due to a stimulation of CDP-choline: ceramide cholinephosphotransferase reaction with subsequent diminution of the rate of phosphatidylcholine synthesis via the CDP-amine pathway (involving cytidine diphosphocholine).