R. Garcea

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.

Cholesterol and phospholipid composition of mitochondria and microsomes isolated from morris hepatoma 51 23 and rat liver

The major component of membranous lipids is represented by phospholipids [l] . It is well known that this class of lipids plays an important role in regulating the biochemical properties of subcellular particles [2] . Moreover, there exists a cholesterol:phospholipid ratio peculiar for several kinds of membranes [3]. Cholesterol is important in stabilizing arrays of phospholipids in cellular and cytoplasmic membranes [4,5] , and in regulating the permeability to small molecules of artificial [6,7] and natural [8] membranes. Lipid-lipid interactions play an important role in the interaction between lipid and protein [3] ; the protein conformation is greatly influenced by their association with the lipid environment [9]. Alterations in the protein components of mitochondrial membranes have been observed in hepatoma mitochondria [ 10,l l] . They could be, at least in part, explained by changes in the lipid composition of membranes. In hepatoma mitochondria, we have also found functional changes, which could be related to high fragility and to an increased resistance to deformation and stretching [ 12,131 . Similar alterations have been observed also in cholesterol-enriched mitochondria [5] . The knowledge of lipid composition of membranes isolated from tumors is lacking and, sometimes, contrasting [ 14,151 . As a first approach to this problem we have studied the phospholipid and cholesterol content of mitochondria and microsomes isolated from rat liver and Morris hepatoma 5 123. 2. Methods

The subcellular distribution and properties of aldehyde dehydrogenase of hepatoma AH-130☆

Aldehyde dehydrogenase subcellular distribution and activity were studied in the Yoshida hepatoma AH-130 and rat liver. NAD+- and NADP+-dependent dehydrogenase activities were lower in all hepatoma subfractions (except the cytosol) than in liver subfractions. In the presence of 0.025 mM substrate 78-80% of the liver NAD+- or NADP+-dependent aldehyde dehydrogenase was found in the mitochondria. With 10 mM substrate the enzyme activity was primarily in the mitochondria and microsomes. In the hepatoma a sharp increase of the soluble aldehyde dehydrogenase (either NAD+- or NADP+ dependent) was observed at all substrate concentrations. The Km of the different isoenzymes (either identified by their localization or coenzyme dependency) were of the same order for liver and hepatoma. However, a high Km enzyme was present in liver mitochondria outer membranes but not in hepatoma. Hepatoma acetaldehyde dehydrogenase was inhibited, as was the liver enzyme, by diethyldithiocarbamate. The return of activity was slower for the hepatoma and neonatal liver than for the adult liver enzyme.

Further Experiments on Lipid Peroxidation in Transplanted and Experimental Hepatomas

The results of experiments on the subject of lipid peroxidation in hepatomas are described. It is now clear that lipid peroxidation is strongly decreased in most highly dedifferentiated hepatomas. It seems evident that the extent of the decline is strictly related to the degree of dedifferentiation. The model of diethylnitrosamine carcinogenesis, according to the method by Soh, Medline and Farber, has been now adopted to study the stages of carcinogenesis. It was shown that a net decline in lipid peroxidation occurs as early as at the stage of reversible nodules and progresses until the development of clear hepatomas. This change is practically simultaneous with a decline in the efficiency of the enzymes of the drug metabolizing system and in the content of cytochrome P450. Glutathione content and metabolism show also important changes. In fact, a dramatic increase in gamma-glutamyl-transpeptidase takes place very early during carcinogenesis, and is responsible for large decline in total glutathione during incubation of the homogenates. Glutathione peroxidase activity, on the contrary, is decreased, whereas glutathione reductase does not show significant changes. The supernatant of highly anaplastic tumors inhibits lipid peroxidation in normal liver homogenates, suggesting the presence of substances provided with antioxidant properties. These cannot be, however, related to a higher glutathione content. Supernatants from early nodules seem to be unable to block lipid peroxidation in normal liver homogenates. Preliminary experiments done to study the aldehyde pattern produced during lipid peroxidation, both in hepatomas and in nodules, confirm the presence of very poor lipid peroxidation and possibly of different peroxidation kinetics.

Modulatory Mechanisms of Chemical Carcinogenesis: The Role of the NADPH Pool in the Benzo(a)pyrene Activation

Human lymphocytes and human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit an 86-87% decrease of this enzymatic activity. This is coupled with 51% and 61% decreases of the NADPH/NADP+ ratio in the G6PD-deficient human lymphocytes (HL) and human skin fibroblasts (HSF), respectively. There also occurs a 63-67% decrease of the hexose monophosphate shunt (HMS) in the deficient cells. Incubation with 0.1 mM methylene blue stimulates the HMS of normal HL 15-fold and that of deficient lymphocytes only 2.4-fold. These figures are, respectively, 7 and 2.2 in the case of HSF. This behavior of G6PD-deficient HL and HSF is coupled with an increase of the resistance to the cell death induced by benzo(a)pyrene (BP). This effect is mimicked by the incubation of normal HSF with dehydroepiandrosterone (DEA) which strongly inhibits G6PD. In contrast, no differences between normal and deficient HSF occur as a result of the effect of methylnitrosourea (MNU), a carcinogen that does not need metabolic activation. The NADPH-cytochrome c (P450) reductase of G6PD-deficient HL and HSF homogenates becomes lower than that of controls when endogenous G6PD and exogenous glucose 6-phosphate (G6P) and NADP+ are used as a hydrogen donor system in place of NADPH. Normal and G6PD-deficient HL, having comparable BP-hydroxylating activities, in the presence of exogenous G6P, NADP+, and G6PD, were studied to determine the effect of the absence of exogenous G6PD in the reaction system. When exogenous G6PD is omitted, the deficient HL exhibit a BP hydroxylase activity sharply lower than controls. G6PD-deficient HL and HSF are less prone than controls to produce BP water-soluble metabolites. A decrease in the synthesis of BP metabolites, mutagenic for his–Salmonella typhimurium in G6PD-deficient HL, has also been observed. These results suggest that the decreased susceptibility of G6PD-deficient cells and HSF to the toxic effect of BP and that of deficient HSF to its previously observed transforming effect, could depend on a reduced BP metabolism in the deficient cells.

Lipid phase transition and breaks in the Arrhenius plots of membrane-bound enzymes in mitochondria from normal rat liver and hepatoma AH-130

The relationships between the physical state of membrane lipids and enzymatic or transport activities have been analyzed in several publications [l-5] using various microorganisms. Discontinuities in the activation energies of enzymatic activities or transport processes were found to depend on the fatty acid composition and cholesterol content of membranes. Such discontinuities sometimes coincided with the phase transition of lipids as detected by X-ray diffraction or thermal analysis [3,5] . However, sometimes disparities between the activity transition temperatures and lipid transition temperatures were observed [ 1 ] . Analogous discrepancies have been seen in studies with isolated mitochondria. Different activities of membrane-bound enzymes of mitochondria exhibit breaks in their Arrhenius plots at 23-24°C and at lo12°C in homeothermic animals and chilling-sensitive plants, respectively [6] . Electron spin resonance experiments showed that the temperatures of the breaks in the Arrhenius plots of some enzymatic activities in rat liver mitochondria coin-

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.

Effect of a cholesterol-rich diet on cholesterol content and phagocytic activity of rat macrophages

Treatment for 11–13 weeks with a cholesterol-rich diet induced increases in free and esterified serum cholesterol. There was also an increase in free cholesterol of peritoneal macrophages. A 2.2 times rise in the cholesterol to phospholipid ratio of plasma membranes occurred in cholesterol-enriched macrophages. No changes were observed in phagolysosomes. Cholesterol-enriched macrophages showed a 35.8% inhibition of latex particles phagocytosis. When lipid droplets were substituted for latex the inhibition was 81.7%.

Phospholipid composition of inner and outer mitochondrial membranes isolated from Yoshida hepatoma AH-130

Outer and inner membranes isolated from hepatoma AH-130 and rat liver mitochondria were used to study phospholipid composition. The phosphatidylethanolamine content increased and that of phosphatidylcholine decreased in ;whole mitochondria and isolated membranes. The ratio between the 2 phospholipids increased 47% and 117% in the inner and outer membranes respectively. A small decrease of diphosphatidylglycerol also occurred in the hepatoma mitochondria inner membrane. In contrast to the results of previous work, no sphingomyelin was found in hepatoma mitochondria and isolated membranes.

Phenobarbital stimulation of cytochrome P-450 and aminopyrine N-demethylase in hyperplastic liver nodules during LD-ethionine carcinogenesis

Microsomes isolated from hyperplastic liver nodules and hepatomas, induced by DL-ethionine, exhibited a reduced cytochrome P-450 content and aminopyrine N-demethylase activity when compared to the organelles of control and surrounding non-nodular liver. Phenobarbital administration to rats caused an increase of microsomal protein, cytochrome P-450 and aminopyrine N-demethylase in all tissue tested. In the hepatoma the rise of cytochrome P-450 and aminopyrine N-demethylase/g of tissue was very low and it is compensated by a slight increase of microsomal protein. In hyperplastic nodules as well as in control and surrounding livers, cytochrome P-450 and aminopyrine N-demethylase increased more than microsomal protein. However, the phenobarbital-induced stimulation was significantly lower in hyperplastic nodules than in control and surrounding livers.