Enzymes metabolizing xenobiotics in spontaneous tumors in mice

The microsomal monooxygenase activity in spontaneous mouse hepatomas has been studied. The cytochrome P-450 level in hepatomas was shown to be 2 times as low as that in the liver. The reduction of the cytochrome P-450 content in the tumour was accompanied by a decrease in the activity of benz(a)pyrene hydroxylase, amino-pyrene-N-demethylase and p-nitroanisole-O-demethylase. However, 7-ethoxycoumarin-O-deethylase activity in hepatomas was much higher than in the liver both estimated as mg of the microsomal protein and nmol of cytochrome P-450. The cytochrome b5 content in the hepatomas was comparable with its level in the liver. A more elevated content of NADPH-cytochrome c reductase and microsomal epoxide hydrolase activity was found in the hepatomas. The results obtained provide evidence of different oxidation effects regarding some substrates in the liver and hepatomas. The ratio of cytochrome P-450 isoforms is likely to change in the hepatomas in contrast with that in the liver.     

Serum lipoproteins in rats bearing Morris hepatomas of different degrees of differentiation.

Serum lipoproteins were measured by ultracentrifugal means in rats bearing hepatomas of different degrees of malignancy (Morris hepatomas 16, 5123TC and 7777) to determine the effect of these hepatomas on serum lipoprotein levels. Serum lipoprotein patterns were altered, especially in rats bearing hepatomas 16 and 7777, which had elevated high-density lipoproteins. (They were not elevated in serum of rats bearing hepatoma 5123TC). This increase in high-density lipoproteins seems to be specific for chemically induced hepatomas since HDL2 is usually decreased in humans and animals with types of cancer not involving the liver. It appears that hepatomas can synthesize lipoproteins, and the serum levels of the host rats are altered depending on the hepatoma. Different biochemistries appear to be associated with each hepatoma. Cholesterol and fatty acid levels of unfractionated serum and of isolated lipoproteins also indicate abnormal lipid/lipoprotein metabolism associated with these hepatomas.     

Content of cytochrome P-450 and its induction capacity in primary liver tumors in rats

The content of cytochrome P-450 has been measured in primary hepatomas induced by diethylnitrosamine. As a rule, the enzyme content in hepatomas was decreased, as compared to normal liver and tumor-affected liver, but some hepatomas contained cytochrome P-450 in greater amount than normal tissue. Aroclor 1254 induced an increase in cytochrome P-450 content, which was identical in hepatomas, normal liver and tumor-affected liver. The dependence of hepatoma morphology on cytochrome P-450 content was not detected.     

Activities of dehydrogenases of the pentose phosphate pathway and transketolase in transplanted mouse hepatomas with different growth rates and in organs of tumor carriers]

The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transketolase were studied in the cytoplasmic fractions of transplanted mouse hepatomas differing in their growth rates, and in the liver, spleen and cortical layer of kidneys of tumour carriers and normal mice. It was shown that transplantation of hepatomas changes the activity of the pentose phosphate pathway enzymes in tumour carrier tissues unaffected by neoplasm. Deviations from normalcy were mainly similar to those observed in the hepatomas. The changes in the enzymatic activities were especially well-pronounced in the mice having rapidly growing hepatomas. This may be due to a generalized effect of the tumour on the organism, which is concurrent with malignancy.     

Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Reduced concentrations of Z protein in Morris hepatomas. Possible role in abnormal regulation of lipid metabolism.

The cytoplasmic concentration of Z protein (Mr approximately 12,000) was significantly reduced in a series of implanted Morris hepatomas with varying degrees of differentiation. Approximately half of the [14C]palmitoyl-CoA added to cytosol fractions from control or host livers was bound to the Z protein region whereas a much smaller proportion was bound to this region in the cytosol of nine Morris hepatomas studied. The possible implications of these findings are discussed in relation to the abnormal regulation of lipid metabolism in hepatomas.     

Comparative study of glycolipid compositions of plasma membranes among two types of rat ascites hepatoma and normal rat liver.

Glycolipid composition of purified plasma membranes from rat ascites hepatomas, two island-forming cell-lines and two cell-lines of the free-type, and normal rat liver were compared. Ceramide monohexoside (CMH), ceramide dihexoside (CDH), and hematoside (GM3) were found in normal rat liver cell membranes. The island-type hepatomas contained ceramide trihexoside (CTh) and globoside besides CMH, CDH, and GM3. The free-type of hepatomas were characterized by the presence of asialo-type gangliosides but not GM3. The free-type of hepatomas were characterized by the presence of asialo-type gangliosides but not GM3. Blood group H active fucolipid was a major glycolipid in the free-type of ascites hepatoma cell (AH 7974 F). The increase of glycolipid content in cell membranes seemed to be accompanied with a decrease of cell adhesiveness.     

dUTP pyrophosphatase and uracil-DNA glycosylase in rat liver and hepatomas.

1. The activity of dUTP pyrophosphatase (dUTPase) was similar in rat liver and hepatomas of slow or moderate growth rate but was increased several fold in three rapidly growing hepatomas. 2. There was an approx three-fold increase in the activity of uracil-DNA glycosylase in Morris hepatoma 7800 but there was little change in activity in other hepatomas that were examined. 3. The activities of dUTPase and uracil-DNA glycosylase were not significantly affected by two diets that may be promotional for hepatocarcinogenesis, a high orotate diet and an arginine-deficient diet.     

Divergent effects of cyanate on amino acid and phosphate uptake by liver and hepatoma.

The uptake of alpha-aminoiso[3H]butyric acid and 32Pi was observed to be inhibited by sodium cyanate in transplanted hepatomas but was increased in the livers of the tumor bearing rats. Incorporation of 32Pi into macromolecules in hepatomas was also inhibited by cyanate. Treatment with this drug did not influence circulating concentrations of isotope-labeled materials. There were relatively small effects on uptake of 36Cl- in cyanate-treated rats and the action was not tissue specific. The data were compatible with an inhibitory effect of cyanate on active transport in hepatomas which was not seen under the same conditions in host liver.     

Cholesterol biosynthesis in transplantable hepatomas: evidence for impairment of uptake and storage of dietary cholesterol

Cholesterol feeding inhibits cholesterol biosynthesis in normal but not in malignant liver tissue. It has been postulated that hepatomas have suffered a specific intracellular deletion of the cholesterol feedback control mechanism, but there is little direct evidence to support this hypothesis. Rats bearing Morris transplantable hepatomas were fed high cholesterol diets for periods of up to 21 days. Cholesterol biosynthesis, as expected, was suppressed in the normal liver but not in hepatomas. The livers accumulated large amounts of cholesteryl ester but the hepatomas showed little or no increase in ester content. Cholesterol-1alpha-(3)H was administered intragastrically to other tumor-bearing rats. Uptake of radioactivity by the tumors was much slower than by normal liver. Comparison of the specific activities of liver and tumor cholesterol with that of the plasma suggested that the liver took up dietary cholesterol selectively from the blood, while the appearance of radioactivity in the tumors could be explained by slow equilibration with plasma cholesterol. Our results suggest that the insensitivity of cholesterol biosynthesis to dietary cholesterol in hepatomas could be explained by an impairment in the uptake and storage of dietary cholesterol and that the concept of an intracellular deletion of the feedback mechanism requires further evidence.     

Regulation of rat liver phosphofructokinase levels in Morris hepatomas.

The levels of the major liver phosphofructokinase isozyme (PFK-L₂) and the PFK regulatory factor were measured in adult and fetal liver as well as Morris hepatomas of different differentiation states. The results indicate that both the PFK-L₂ activity and the PFK regulatory factor levels in well and highly differentiated hepatomas are not statistically different from the amounts found in adult liver. In the poorly differentiated hepatomas and fetal liver, the levels of both PFK-L₂ and PFK regulatory factor are approximately 2 and 3 fold greater, respectively, than what was found in adult liver.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

Negative correlation of L-glutamine concentration with proliferation rate in rat hepatomas

The concentration of L-glutamine was determined in freeze-clamped samples of normal liver of adult male fed rats (5.7-6.1 mumol/g) and in transplantable hepatomas of vastly different proliferative rates. The L-glutamine concentration in the slowly growing hepatomas was in the range of the normal liver and it decreased in relation to the increase of hepatoma growth rate, in the most rapidly growing tumors amounting to 12% of that of normal liver. In 24-hour regenerating liver, the glutamine content was slightly reduced (by 17%). In normal rat organs of high cell renewal, such as testis, intestinal mucosa, spleen, and thymus, the L-glutamine concentration was 18 to 46% of that of normal rat liver. The L-glutamine content was similar in rat brain and liver, but it was 1.6-fold higher in the heart, and low in the blood. Glutamine synthetase (EC 6.3. 1.3) activity in normal adult liver of ACI/N strain rats was 1,000 nmol per hr per mg protein; the activity increased in the very slowly growing hepatoma 20, but decreased markedly in all the other hepatomas. Thus, glutamine synthetase activity was essentially transformation-linked. The negative correlation of glutamine content with growth rate in transplanted hepatomas appears to be more closely linked with the activities of enzymes that utilize glutamine. The low L-glutamine concentration in the rapidly growing hepatomas provides a potential marker for anti-glutamine chemotherapy selectively targeted against the glutamine-utilizing enzymes.     

Gangliosides depleted in plasma membrane are directed to internal membranes of rat hepatomas: evidence for a glycolipid sorting defect in hepatocarcinogenesis

Ganglioside compositions of plasma membrane fractions highly purified from rat liver and hepatomas by phase partitioning were compared with those of fractions composed of internal membranes, free of plasma membrane. With liver, 70-80% of the the lipid bound sialic acid were accounted for by a plasma membrane location. In hepatomas this percentage was reduced to 50-65%. More pronounced was the distribution of the simple monosialoganglioside GM3. In the hepatomas, 60-80% of the GM3 was found associated with internal membranes as compared to liver where only 35% of the GM3 was present in internal membranes. The findings suggest a glycolipid sorting defect in hepatocarcinogenesis where gangliosides, and especially monosialogangliosides, are diverted to internal membranes rather than being correctly transported to the cell surface. Since GM3 is synthesized exclusively in the Golgi apparatus of both liver and hepatomas, the basis for the sorting defect may reside in a functionally altered Golgi apparatus.     

Properties of the inactive ribosomal components in rat liver and hepatoma

1. The relative concentrations of the inactive ribosomal components were compared in normal and regenerating rat liver and in two transplantable rat hepatomas (hepatomas 7800 and 5123D). 2. The size of the ribosomal-subunit pools in normal liver was not significantly affected by partial hepatectomy or neoplasia although, as shown previously, significant changes do occur in the monomer pool. 3. Further, the subunit pools in both liver and hepatoma were not significantly influenced by several treatments that caused dramatic changes in the size of the ribosomal monomer (and dimer) pools. 4. The high concentration of inactive monomers and dimers in the hepatomas appears to arise from limitations at the translational level, since they can be incorporated into pre-existing polyribosomes under the influence of cycloheximide.     

Some characteristics of isoenzymic spectrum of hexokinase and glucose levels in hepatomas and liver of the host]

The total activity of hexokinase (HK) and HK isoenzymic spectrum of the normal liver and slowly groming hepatoma 49 did not show any essential differences. However, the HK total activity and the relative and absolute contents of isoenzyme HK-3 were increased in hepatomas 61 and especially in the rapidly growing hepatoma 22-a. The glucokinase activity decreases in the hepatiomas 49 and 61 and disappears in the rapidly growing hepatoma 22-a. The glucose content in hepatoma 49 was slightly lower than in the normal liver, whereas in other hepatoma no traces of glucose could be detected. At low glucose concentration in the medium (0,1 mM), i.e. under conditions simulating those characteristic of tumors in vivo, the predominant form of HK in all hepatomas studied was found to be HK-3. In the liver of hepatoma-bearing mice some shifts in the value of total HK activity and its isoenzymic spectrum, reminding one of those found in hepatomas themselves, were observed. Unequal deviations in the total HK activity and its isoenzymic spectrum in hepatomas with different degrees of malignancy indicate that these characteristics are secondary rather than primary events depending on tumour progression.     

DNA content in cells of spontaneous hepatomas in CBA mice

The DNA content was measured in the cells of 37 well-differentiated spontaneous hepatomas from 20 CBA mice aged 18 to 22 months. In all the cases, the DNA distribution pattern was polymodal, with the class of tetraploid cells being predominant. It was independent of the tumor ability to produce AFP. No signs of aneuploidy were noted. The cells of spontaneous hepatomas were characterized by a high level of polyploidy and by a dramatic reduction in the number of binuclear cells as compared with liver cells. A tendency was recorded toward positive correlation between the degree of cellular polyploidy and tumor size. The high level of polyploidy seen in spontaneous hepatomas which is also characteristic of the normal liver is considered to be a sign of tumor maturity.     

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : IV. Some Observations on the Properties of Ribosomes and Polysomes from Rat Liver and Hepatomas

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min'' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.     

Chromosomal nonhistone proteins of rat hepatomas and normal rat liver

Chromatin isolated from several well-differentiated rat hepatomas and regenerating liver contains more nonhistone proteins than chromatin of normal liver. Also there are several differences in the electrophoretic patterns of chromosomal nonhistone proteins between hepatomas and normal liver, but not between regenerating or fetal liver and normal liver.