Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.

BACKGROUND: Alkylphosphocholines (APCs) are synthetic phospholipid derivatives, and have been demonstrated to inhibit ocular cell proliferation in vitro and in vivo. Currently, they are applied clinically for their antitumoral and antiparasitic properties, but have not yet been implemented for clinical use in proliferative ophthalmic disorders. The purpose of this study was to assess the safety of APC in the ex vivo model of the isolated perfused vertebrate retina. METHODS: Bovine retina preparations were perfused with an oxygen pre-equilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl-electrodes. After recording stable b-wave amplitudes, an APC was applied at the following concentrations to the nutrient solution: 0.25 microM, 2.5 microM and 25 microM. To investigate the effects of APC on photoreceptor function, a test series at the same concentrations was performed to evaluate the effects of APC on the a-wave amplitude. Aspartate at a concentration of 1 mM was added to the nutrient solution to obtain stable a-wave amplitudes. Thereafter, APC was applied at the same concentrations to the nutrient solution. The recovery of the ERG amplitudes was followed up for 75 minutes. RESULTS: No reduction of the a- and b-wave amplitude was found at the end of the exposure time with APC added in each test series. No differences were found between the ERG amplitudes before and after application of APC at the end of the washout. CONCLUSIONS: In the ex vivo model of the isolated perfused vertebrate retina, APC has proved to be a safe compound in the concentrations applied. Thus, APCs should further be considered as promising candidates for future clinical applications in ophthalmology.

Is dietary erucic acid hepatotoxic in pregnancy? An experimental study in rats and hamsters.

The hypothesis that dietary erucic acid may contribute to the pathogenesis of intrahepatic cholestasis of pregnancy has been examined in pregnant rats and hamsters after prolonged feeding of diets containing 25% rapeseed oil rich in erucic acid (40% of fatty acids) or corn oil, without erucic acid. Both dietary oils were well tolerated, although weight gain was 17% to 20% less in animals receiving rapeseed oil. Rats and hamsters were studied on the last day of pregnancy and compared with age- and diet-matched nonpregnant animals. Histological examination showed no major morphologic abnormalities in liver, heart, kidneys, and adrenals. Similar microscopic deposits of fat were found in the livers and hearts of pregnant hamsters of both dietary groups. Chromatographic analysis of fatty acids in liver, heart, and kidney homogenates of hamsters and in isolated rat liver cells reflected the fatty acid composition of the dietary oils: oleic (18:1) and linoleic (18:2) acids were among the predominant fatty acids. Erucic acid was found in a higher proportion in the heart (14% by weight of total fatty acids) than in the liver (3%) and kidneys (3%) of animals fed rapeseed oil. Bile flow and biliary lipid composition was similar in rats and hamsters fed rapeseed or corn oil. Bile flow tended to be less in pregnant than in nonpregnant animals. Pregnant hamsters fed rapeseed oil tended to have the lowest bile flow. The lithogenic index of bile was slightly decreased in pregnant rats and increased in pregnant hamsters, although these proportional changes were similar for both diets. In all circumstances the lithogenic index remained below a value of 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and toxicological studies on the effect of high and low erucic acid rapeseed oil on rats.

Rats were fed on diets containing rapeseed oil, either containing low or high erucic acid content as well as the hydrogenated ones for 6 weeks. Body weight gain, biochemical and pathological parameters were investigated. The data showed high body weight gain for rats fed diets containing low erucic rape oil (LERo) compared with those fed either the high erucic rape oil (HERo), the hydrogenated or the partially hydrogenated oil diets. All rats showed non significant changes for total lipids, total cholesterol, GPT and GOT, except the partially hydrogenated rape oil diet which showed significant decrease for total cholesterol. Alkaline phosphatase however showed a significant decrease, while plasma phospholipids showed significant increase in rats fed on the hydrogenated oil diet. Triglycerides indicated non significant increase except in the group that received low erucic rape oil diet. Histopathological study showed changes in all tissues examined (liver and kidney).

Influence of dietary rapeseed oil and erucic acid upon myocardial performance and hemodynamics in rats.

Rats fed rapeseed oil, pure erucic acid, or a control diet of sunflowerseed oil during 24-26 weeks were studied for effects upon mechanical behavior of the isolated perfused heart and upon myocardial performance and hemodynamics in intact animals both under basal and stimulated conditions. In spite of focal myocardial fibrotic lesions due to rapeseed oil, no changes were found with respect to the intrinsic myocardial contractility in vitro and and in vivo. After inotropic intervention, only the rapeseed oil fed animals showed less contractile reserve capacity. The absence of this effect in the erucic acid-treated animals is in agreement with the histological studies showing no epicardiac fibrotic lesions in these animals. It appears that erucic acid is able to interfere with the contractile system of the peripheral vascular system. Both in the rapeseed oil-treated group and the erucic acid-treated group, the vasoconstrictor response toward norepinephrine was profoundly reduced. In all three oil fed groups, isoproterenol reduced myocardial contractility which has been attributed to a lowered perfusion pressure in the coronary blood supply of the myocardium with simultaneous increased energy demand. Neither rapeseed oil nor erucic acid feeding led to electrocardiographic changes in comparison with the control sunflowerseed oil group. It is concluded that rapeseed oil and not erucic acid is responsible for loss of contractile reserve capacity without changes in the myocardial conductance system and further, that erucic acid might interfere with the peripheral vascular system. Finally, it appears that a fat rich diet might result in reduced myocardial function during a state of energy demand coupled with a blood pressure decrease.

Metabolism of very long-chain monounsaturated fatty acids (22:1) and the adaptation to their presence in the diet.

Unadapted rats and other animal species have a limited capacity to metabolize monounsaturated fatty acids with 22 carbons (22:1). Excess amounts in the diet of fats containing these fatty acids cause a transient accumulation (lipidosis) of triacylglycerol in the heart and other tissues but not in the liver, which seems to export the 22:1 fatty acids as very low density lipoproteins to the blood plasma. The acute lipidosis most probably is explained by a slow oxidation of 22:1 acyl-CoA by the mitochondrial acyl-CoA dehydrogenase combined with an inhibitory effect of this CoA ester on the oxidation of acyl-CoA esters of a more "normal" chain length. Other fatty acid metabolizing enzymes also show slow reaction rates with the 22:1 fatty acids. Upon continued feeding of diets with 22:1 fatty acids, an adaptation takes place and the lipidosis disappears. This adaptation coincides with the development of an increased capacity to chain-shorten the 22:1 fatty acids, especially in the liver, but also in the heart. The chain-shortening seems to be due to a partial beta-oxidation of the 22:1 fatty acids by the peroxisomal beta-oxidation enzyme system which shows an increased activity in adapted rats. In such rats, less 22:1 fatty acids circulate in the plasma very low density lipoproteins than in unadapted rats. The drug clofibrate (ethyl-p-chlorophenoxyisobutyrate) which induces increased activity of the peroxisomal beta-oxidation enzymes, provides partial protection against the lipidosis in unadapted animals. Hydrogenated fish oil (containing different 22:1 isomers and many fatty acids with trans double bonds) is more efficient as an inducer of the chain-shortening of erucic acid in the liver than is rapeseed oil, which contains only one 22:1 fatty acid isomer and no fatty acids with trans double bonds. The hydrogenated fish oil causes less lipidosis than does rapeseed oil when diets containing the same amount of 22:1 fatty acids are fed. It is suggested that CoA esters that are poorly oxidized by the mitochondria (e.g., esters of erucic acid, of some fatty acids with trans double bonds, and of clofibric acid) may trigger the adaptation process.-Bremer, J., and K. R. Norum. Metabolism of very long-chain monounsaturated fatty acids (22:1) and the adaptation to their presence in the diet.

Long-term effects of partially hydrogenated herring oil on the rat myocardium.

Male Sprague-Dawley rats were fed semisynthetic diets containing 25% (w/w) experimental lipid. A partially hydrogenated herring oil (PHHO), containing 25% C22:1 as an isomeric mixture dominated by cetoleic (cis-docos-11-enoic), but partially altered to the trans form and containing other positional isomers, was compared with lard:corn oil 3:1(LCO) (w/w). The feeding period extended up to 111 weeks. Ten animals from both groups each were sacrificed at 2, 8, 16, 24, and 32 weeks. The remaining animals from each group were kept on the diets either until spontaneous death occurred or until Week 111. The LCO rats had higher serum cholesterol levels. The PHHO rats had transient myocardial lipidosis and very severe, progressive myocardial inflammation and scarring.

Morphologic effects of dietary plant and animal lipids rich in docosenoic acids on heart and skeletal muscle of cynomolgus monkeys.

Cynomolgi (Macaca fascicularis) were fed diets containing 25% rapeseed oil (RSO), partially hydrogenated herring oil (PHHO), or a 3:1 mixture of lard and corn oil as control for 4 months. The RSO contained approximately 25% of the fatty acids as erucic acid; the PHHO contained a similar concentration of mainly cetoleic acid. The control diet did not include such fatty acids. At the time of necropsy, the RSO- and PHHO-fed monkeys showed myocardial and skeletal muscle lipidosis. Foci of mononuclear cell infiltration, although infrequent, occurred in all three groups and were thought to be nonspecific. The only significant intergroup difference in serum biochemical or hematologic parameters was an increase in serum glutamic-oxaloacetic transaminase activity in both RSO and PHHO groups. Ultrastructural studies confirmed the presence of lipidosis in cardiac and skeletal muscle and revealed mild mitochondrial degeneration, causing a depression of the P/O ratio of the RSO group and a State III respiratory rate depression of the PHHO group. The difference in the exposure/life span ratio represented by this experiment may account for the absence of clear intergroup differences such as are reported in rats used in similar studies, but a true species difference in regard to dietary oils containing docosenoic acids has to be considered as well.

Biochemical and histological effects of feeding thermally oxidized rapeseed oil and lard to rats.

Four groups of weanling rats were fed semisynthetic diets containing 15 percent by weight of dietary fats for 28 days. Two groups received thermally oxidized low-erucic acid rapeseed oil (OLE) or lard (OLA) and the other two groups received the respective fresh fats (FLE, FLA) as controls. Average daily feed consumption and feed efficiency were not affected by either OLE or OLA, however final body weights were depressed by the OLA in the diet. The relative heart weights and heart total lipids were significantly increased in both the OLE and OLA groups. A majority of the animals in both the OLE and OLA groups exhibited various gross symptoms attributable to heated fat toxicity such as seborrhea, diarrhea and polyuria. Excessive hair loss was noted in all the animals fed the heated fats. Tissue fatty acid changes due to OLA were confined largely to the polar liver lipids, whereas OLE produced dramatic changes in both the neutral heart lipids as well as in the neutral and polar liver lipids. Histological evaluation of the hearts, livers and kidneys indicated that OLA was very injurious to the kidneys, whereas OLE caused greater damage to both the hearts and livers.

Mortality and production characteristics of laying chickens fed high- and low-erucic acid rapeseed oils.

Laying pullets were fed a diet supplemented with three percent of either high-erucic acid rapeseed (HEAR) oil or low-erucic acid rapeseed (LEAR) oil for 39 weeks. Egg production for the period was 78.8 and 80.1% by the birds fed the respective oils. Average egg weights were 56.8 and 58.7 g. respectively. Gains in body weight, liver weight and adrenal gland weight were similar with the two types of oil fed. Feed consumption was similar for the two groups of birds. The efficiency of utilization of the diet containing HEAR oil was accordingly lower than that of the diet containing LEAR oil. In the birds fed HEAR and LEAR oil respectively mortality attributed to metabolic disorders of the reproductive system, liver, and kidney was 3.3% and 1.2%. Mortality from other causes in the birds fed the two oils was 3.2 and 3.8% of the original populations.

Investigations on rapeseed oil toxicology.

A series of investigations on humans and mice, performed in this laboratory in the years 1973-74 is presented. To assess the exposure levels of the Italian population to rapeseed oil ingestion, the serum of 70 apparently healthy railway workers, as well as the adipose tissue and myocardium of 16 subjects deceased for different causes were analyzed, with a gas chromatographic method, to ascertain if erucic acid or its metabolites were present. 40 out of 70 subjects had measurable erucic acid in the serum ranging from 0.3 to 3.8% of the total fatty acids (mean 1.1 +/- 0.14) in the presence or absence of behenic acid, probable metabolite of erucic acid in humans. Furthermore 26 subjects had measurable behenic acid alone. Each of the 16 subjects had measurable erucic acid in adipose tissue ranging from 0.3 to 6.8% of the total fatty acids (mean 2.6 +/- 0.5) always in the presence of gadoleic acid (present in rapeseed oil, product of beta-oxidation of erucic acid and only partially deriving from other sources) ranging from 1.1 to 4.9% (mean 2.2 +/- 0.3). 13 subjects out of 14 analyzed had measurable erucic acid in the myocardium ranging from 0.2 to 2.2% (mean 0.7 +/- 0.18) while 9 subjects had measurable gadoleic acid. Erucic acid was absent in the human tissues in Italy in the years 1967-68. Regarding the studies on mice, the deposition of erucic and gadoleic acids in some organs of young mice supplied with 50% of daily calories for three days in rapeseed oil was investigated. The deposition of the two fatty acids amounts to about 10% of the total fatty acids in the heart and liver and considerably less in skeletal muscle and kidney. In the mouse liver there is also a decrease in hexobarbital metabolism following rapeseed oil ingestion.

Physiopathological effects of rapeseed oil: a review.

Rapeseed oil has a growth retarding effect in animals. Some investigators claim that the high content of erucic acid in rapeseed oil alone causes this effect, while others consider the low ratio saturated/monounsaturated fatty acids in rapeseed oil to be a contributory factor. Normally erucic acid is not found or occurs in traces in body fat, but when the diet contains rapeseed oil erucic acid is found in depot fat, organ fat and milk fat. Erucic acid is metabolized in vivo to oleic acid. The effects of rapeseed oil on reproduction and adrenals, testes, ovaries, liver, spleen, kidneys, blood, heart and skeletal muscles have been investigated. Fatty infiltration in the heart muscle cells has been observed in the species investigated. In long-term experiments in rats erucic acid produces fibrosis of the myocardium. Erucic acid lowers the respiratory capacity of the heart mitochondria. The reduction of respiratory capacity is roughly proportional to the content of erucic acid in the diet, and diminishes on continued administration of erucic acid. The lifespan of rats is the same on corn oil, soybean oil, coconut oil, whale oil and rapeseed oil diet. Rats fed a diet with erucic acid or other docosenoic acids showed a lowered tolerance to cold stress (+4 degrees C). In Sweden erucic acid constituted 3-4% of the average intake of calories up to 1970 compared with about 0.4% at present.

Morphological effects of rapeseed oil in rats. I. Short-term studies.

Light microscopy of paraffin embedded and frozen sections, supplemented with electron microscopy, was performed on the heart muscle of young rats fed rapeseed oil in short-term experiments. It was confirmed that high levels of rapeseed oil, which contains erucic acid, produce severe lipoidosis of the heart muscle fibres within 10 days. An attempt was made to find out the lowest level of erucic acid in the rat diet to give rise to pathological fatty accumulation. Several frozen sections from each heart or serial sections in combination with electron microscopy were used for this evaluation. The level found to give rise to pathological fatty accumulation was about 2% by weight (w/w), while rats fed 1% erucic acid showed normal myocardium. No direct proof that erucic acid is of importance in human pathophysiology has hitherto been presented. It is concluded, however, that the similarity in reaction among the many species of experimental animals tested by different workers, as well as the basic metabolic disturbances demonstrated, are in strong favour of a similar effect in man.

Morphological effects of rapeseed oil in rats. II. Long-term studies.

In long-term studies covering up to 160 days young Sprague-Dawley rats were fed diets containing 40 cal% of fat. The fat component consisted of either conventional rapeseed oil, or Canadian rapeseed oil low in erucic acid, or arachis oil. Myocardial fatty accumulation was demonstrated in light microscopic studies throughout the experiments in rats fed conventional rapeseed oil, but the number of fat droplets decreased with time. The controls fed arachis oil showed no fatty accumulation. In the rats fed conventional rapeseed oil focal myocardial lesions appeared after 40 days on the diet. These consisted of histiocytic infiltration, occurrence of macrophages, myolysis, proliferation of fibroblasts and finally scarring. Such foci were found widely spread in the myocardium of these rats. In the experimental groups given Canadian rapeseed oil from the cultivar Oro no histiocytic foci or scarring were observed. Small myocardial lesions were occasionally found in the control rats. These latter findings were observed on serial sections. It was concluded that this type of lesion is a "normal" finding. The number and size of the foci observed in animals fed conventional rapeseed oil (10% and 2% (w/w) erucic acid in the diet) indicate, however, that they have to be considered pathological under such circumstances. The pathogenesis of the myocardial alteration is discussed and it is concluded that the long-chain fatty acids are responsible. No direct proof has been presented that the described events are of importance in human pathophysiology, However, several circumstances pointing in this direction are discussed. It is concluded that on the basis of our present knowledge a pathological effect of erucic acid and its homologues in man cannot be excluded.

Morphological effects of rapeseed oil in rats. III. Studies in germ-free rats.

The morphological effects on the myocardium of feeding rapeseed oil were compared in conventional and germ-free rats in short-term experiments (10 days). It was concluded that the fatty accumulation in the heart muscle cells occurring in rats fed rapeseed oil was not influenced by the presence or absence of a normal intestinal flora. In long-term experiments (80 days) under similar conditions, the myocardial effects of feeding germ-free rats with conventional rapeseed oil, rapeseed oil from the Canadian cultivar Oro very low in erucic acid, or arachis oil were studied in serial sections. Severe myocardial lesions developed in the group of rats fed conventional rapeseed oil, while in the other two groups the myocardium was completely normal. These results give no support to the theory that other factors than C22:1 acids in rapeseed oil are responsible for the myocardial lesions.

Electrocardiogram and renal concentrating capacity in rats fed a diet containing rapeseed oil.

The functional effects of a diet containing rapeseed oil (40% of total energy intake) were studied in rats, starting at age 28 days. There were no effects on the electrocardiogram in spite of morphological changes in the myocardium. In 10 female rats the urine osmolality following 16 hours of dehydration was approximately 20% lower than in 10 control rats during the 9th, 10th and 20th week of the experiment. It is suggested that erucic acid in the rapeseed oil inhibits beta-oxidation of fatty acids in the kidney, thereby depriving the kidney of energy involved in sodium transport.