Functional alterations of the endoplasmic reticulum and the detoxification systems during diethyl-nitrosamine carcinogenesis in rat liver

The mixed-function oxidase system shows a number of variations in the liver of diethyl-nitrosamine (DEN) treated rats. These include a decrease of the cytochrome P450 content and of the aminopyrine demethylase activity both in the hyperplastic nodules and in the hepatoma. Processes of detoxification, such as the glutathione system, show some modifications. These alterations are in accordance with the decrease of glutathione peroxidase and the increase of gamma-glutamyltranspeptidase during diethyl-nitrosamine carcinogenesis.     

Lipid composition of the plasma membrane isolated from hyperplastic nodules of rat liver

Hyperplastic nodules and hepatomas were induced in livers of rats fed a diet containing 0.05% N-2-fluorenylacetamide (2-FAA). The lipid contents, and phospholipid and fatty acid compositions were analyzed in plasma membranes (PM's) isolated from these tissues and normal rat liver, and the following trends were observed. The molar ratio of cholesterol to phospholipid-phosphorus (phospholipid-P) increased in the order: hepatoma less than normal liver less than hyperplastic nodules. The molar percentage of plasmalogen to phospholipid-P decreased in the order: hepatoma = hyperplastic nodules greater than normal liver. The percentages of choline phosphoglycerides (sum of phosphatidylcholine and lysophosphatidylcholine) and ethanolamine phosphoglycerides (sum of phosphatidylethanolamine and lysophosphatidylethanolamine) both decreased in the order: hepatoma greater than hyperplastic nodules greater than normal liver. On the other hand, the percentages of sphingomyelin and phosphatidylserine both increased in the order: hepatoma less than hyperplastic nodules less than normal liver. As regards fatty acid composition, the percentages of both 18:1 and 18:2 decreased in the order: hepatoma greater than hyperplastic nodules greater than normal liver. Those of 18:0 and 20:4 increased in the order: hepatoma less than hyperplastic nodules less than normal liver. These results suggested that the lipid bilayer in PM of hyperplastic nodules has characteristics roughly intermediate between those of hepatoma and liver PM's, although the molar ratio of cholesterol to phospholipid-P in hyperplastic nodules PM was not intermediate.     

Effect of dietary vitamin E on methyl ethyl ketone peroxide damage to microsomal cytochrome P-450 peroxidase

The effect of dietary vitamin E on in vivo and in vitro damage by methyl ethyl ketone peroxide (MEKP) to cytochrome P-450 and its associated enzymatic activity was studied. In vivo, MEKP damaged microsomal cytochrome P-450 and cytochrome P-450-mediated peroxidases in vitamin E-deficient rat liver. Dietary vitamin E treatment of rats protected the microsomal enzymes from peroxide damage. In vitro, the extent of MEKP inhibition was different for tetramethylphenylenediamine (TMPD)-peroxidase, NADH-peroxidase, and aminopyrine demethylase. In vitro addition of MEKP induced production of more thiobarbituric acid reacting substances (TBARS) in liver microsomes from vitamin E-deficient rats than from vitamin E-supplemented rats. When NADH and/or NADPH were supplied as reductants of MEKP, the inhibition of aminopyrine demethylase activity and the generation of TBARS by added MEKP were markedly reduced. In vivo, adequate levels of vitamin E and of NADH and NADPH are probably necessary to provide important protection to the endoplasmic reticulum during metabolism of toxic organic peroxides, such as MEKP.     

High expression of an N-acetylglucosaminyltransferase III in 3'-methyl DAB-induced hepatoma and ascites hepatoma

Ascites hepatoma AH-66 and 3'-Me-DAB-induced hepatoma of rats contain highly active N-acetylglucosaminyltransferase III (GnT-III) which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core of asparagine-linked sugar chains, whereas normal rat liver contains very little. The high activity was also detected in fetal rat liver, newborn rat liver, hyperplastic nodules and various transplantable hepatomas.     

Effects of phorone (diisopropylidene acetone), a glutathione (GSH) depletor, on hepatic enzymes involved in drug and heme metabolism in rats: evidence that phorone is a potent inducer of heme oxygenase

Concomitant with the depletion of glutathione content, phorone (250 mg/kg, ip.) produced a marked increase in heme oxygenase activity, biphasic effect on delta-aminolevulinic acid synthetase activity, and slight decreases in cytochrome P-450 content and aminopyrine demethylase activity in the liver of rats. The increase in heme oxygenase activity evoked by phorone was almost completely blocked by pretreatment of rats with actinomycin D and cycloheximide. Phorone was able to produce the changes in these parameters in a dose-dependent manner. Buthionine sulfoximine, a GSH depletor by inhibition of biosynthesis, failed to affect these hepatic parameters.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

Resistance to oxidative stress by hyperplastic and neoplastic rat liver tissue monitored in terms of production of unpolar and medium polar carbonyls

The susceptibility of rat liver tissue to oxidative stress during its neoplastic transformation was analyzed by both qualitative and quantitative measurements of the carbonyl products of lipid peroxidation. Diethylnitrosamine was used as initiating agent of hepatocarcinogenesis and lipid peroxidation levels were monitored in the homogenates from normal liver, hyperplastic nodules and tumour, incubated in the presence or in the absence of ascorbate or adenosine diphosphate-iron complex. While the basal levels of lipid peroxidation in the three experimental conditions were found to be quite similar, in the presence of the pro-oxidant stimulus a remarkable reduction in aldehyde production was shown not only by the hepatoma tissue but also by the preneoplastic nodules.     

Rapidly labelled ribonucleic acid from rat liver. Studies in the attachment to ribosomes and stimulation of the incorporation of amino acids into polypeptides in cell-free systems

1. Rapidly labelled RNA from rat liver, either as a complex with DNA (m-RNA-DNA) or with ribosomal RNA (m-RNA-RNA) binds to ribosomes in the polysome region. No binding could be demonstrated with ribosomal RNA or native DNA from Bacillus subtilis. 2. With ribosomes from rat liver, Escherichia coli or hepatoma the m-RNA-DNA stimulated incorporation of amino acids with rat-liver ribosomes only, whereas the m-RNA-RNA complex was effective with ribosomes from E. coli or the hepatoma. 3. Polyuridylic acid was effective as messenger RNA with all three ribosomes but much greater stimulation was obtained with ribosomes from E. coli and the hepatoma. 4. The degree of incorporation of phenylalanine with polyuridylic acid and ribosomes from a hepatoma was decreased by about 50% when ribosomal RNA was present.     

Mechanisms of alpha-fetoprotein synthesis in hepatoma cells. Combined electron microscopic immunocytochemistry and autoradiography

Immunoreaction of alpha-fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP. Combined light microscopic immunoperoxidase study and autoradiography with 3H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

Proliferative response of putative preneoplastic hepatocytes to triiodothyronine, aminoacids, glucagon and heparine

Proliferative advantage of putative preneoplastic hepatocytes measured as the ratio of tritiated thymidine incorporation into cells of hyperplastic nodules with respect to the incorporation in hepatocytes of extranodular liver has been found to be about 2 in rats treated according to 4 carcinogenesis protocols consisting in one or two cycles of diethylnitrosamine and phenobarbital administration. The proliferative response of hepatocytes in hyperplastic nodules to the intravenous infusion of triiodothyronine, amino-acids, glucagon, and heparine (TAGH) has been found higher or similar--except in one case--than the response of surrounding liver but the proliferative advantage of preneoplastic hepatocytes is lower after TAGH stimulation than in basal conditions in all cases.     

Induction of cytochrome(s) P450-dependent drug metabolism in cultured MH1C1 hepatoma cells

A cell line derived from a Morris hepatoma, MH1C1, was examined for its in vitro expression of monooxygenases. These cells were found to contain different forms of cytochrome P450, as shown by the response to inducers, namely phenobarbital (PB), 3-methylcholanthrene (MC) and metyrapone (MP). MH1C1 cell monolayers exposed to PB or MC showed an increase in the concentration of two spectrally distinct forms of cytochrome P450. The PB and MC treatments elicited enzyme activities towards the substrates aminopyrine and benzo(a)pyrene, respectively. The cell treatment with metyrapone led to a simultaneous stimulation of aminopyrine demethylase and benzo(a)pyrene hydroxylase activities, so underlining the peculiar features of this inducer.     

Correlation of membrane-bound and free ribosomes in normal rat liver, Zajdela hepatoma rat liver and ascite cells proper]

The content of membrane-bound ribosomes in normal rat liver cells is 3 times as high as compared to that of free ribosomes. (K=membrane-bound ribosome RNAs divided by free ribosome RNAs=3, the opposite effect being observed in case of ascites hepatoma cells. A considerable increase in the free ribosome fraction in the liver of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of hepatoma-bearing rats occurs by the sixth day due to a decrease in the content of membrane-bound ribosomes (K=0.6). Similar, but less-pronounced changes were observed in liver cells of control animals after 48-hour starvation (K=0.9), simulating the condition occurring during the last days of tumour animals' life. Thus, changes in the rativ of membrane-bound to free ribosomes in liver during the ascites tumour growth are probably specifics and are not only due to anorexia in Zajdela hepatoma animals.     

Mechanisms of α-fetoprotein synthesis in hepatoma cells

Summary Immunoreaction of -fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP.Combined light microscopic immunoperoxidase study and autoradiography with ³H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

The microsomal oxidation system of the liver in the aging of animals with various specific lifespans

Experiments, carried out on adult and old animals with different specific lifespan (rat, guinea pig, rabbit, and dog), revealed age-related changes in content and activity of liver microsomal oxidation enzymes (cytochrome P-450, b5, aminopyrine demethylase, aniline hydroxylase). The changes become more pronounced with age. This allows to classify these species as chronobiological ones. A negative correlation between the specific lifespan and the level of decrease in activity of aminopyridine demethylase, an integral index of electron transport rate in microsomal chain, in aged animals was established.     

Early biochemical alterations induced by 2-acetylaminofluorene in rat liver

Livers from rats fed the carcinogen 2-acetylaminofluorene (AAF) were analyzed at weekly or semiweekly intervals to correlate appearance of enzymatic markers in total liver homogenates with histochemical events accompanying formation of hyperplastic liver nodules. gamma-Glutamyltranspeptidase (gamma-GT)-positive foci appeared by day 11 and visible nodules were present by days 28-35. Specific activity of homogenate gamma-GT increased in parallel to formation of hyperplastic foci and nodules, declined and then rose again to 20-fold that of controls by day 77. Specific activity of ornithine decarboxylase increased in advance of that of gamma-GT, to a level of 8-fold above control during the period of formation of hyperplastic foci. An early response was a 2-fold rise in the specific activity of nucleoside diphosphate phosphatase during the first week of carcinogen administration. The specific activity of 5'-nucleotidase, known to increase during liver regeneration, declined as the animals aged and was not increased by the dietary AAF. The enzymatic alterations induced by AAF could not be mimicked by cell proliferation, diet stress or the hepatotoxicity induced by feeding 1.87% 4-acetamidophenol.     

Hyperplastic foci in chemical carcinogenesis]

Morphological, histochemical and biochemical perculiarities of liver hyperplastic nodules induced by different chemical carcinogens are regarded. The presence of basophylic atypical cells in the hyperplastic nodules, the possibility of transplantation of some nodules, the irreversibility of some morphological and biochemical lesions in these allows to designate hyperplastic nodules as a neoplasm. Enzyme and isozyme patterns in hyperplastic vesicles, the appearance of alpha-fetoprotein in these, and some other metabolic findings suggest biochemical disdifferentiation occurring in the vesicles. These findings suggest that cells of hyperplastic nodules are intermediate between normal and malignant cells in the course of neoplastic transformation.     

Stimulation of hepatic cytochrome b5-mediated lipid desaturation by renal microsomes

Although microsomes prepared from rat kidney cortex contained significant concentrations of both NADH cytochrome b₅ reductase and cytochrome b₅, they did not catalyze cytochrome b₅-dependent Δ⁹ oxidative lipid desaturation. However, incubation of kidney microsomes in the presence of control liver microsomes resulted in a two-fold increase in fatty acid desaturase activity over that seen with liver microsomes alone. Addition of kidney microsomes to liver microsomes prepared from animals maintained on a fat free diet resulted in an increased desaturase activity which was twice that seen with the control liver preparation. Kidney microsomes alone did not catalyze the cytochrome P-450-dependent N-demethylation of aminopyrine, and in contrast to the desaturate, no increase in demethylase activity was observed when kidney microsomes were added to liver microsomes.     

Decrease of the activity of the mixed function oxidase system in regenerating rat liver: An alternative explanation

Partial hepatectomy and sham-operation reduce to a similar extent the activity of two enzymes of rat liver mixed function oxidase system, aniline hydroxylase and aminopyrine demethylase. On this basis it is proposed that the decreased functional specialization of regenerating liver depends on events associated to the stress of intra-abdominal surgery and not, as previously hypotized, to cellular multiplication. This view is confirmed by the fact that regenerating liver retains its ability to respond to phenobarbitone administration with an increased activity of the two enzymes.     

Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid

The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).     

gamma-Glutamyl transpeptidase in rat liver during 3'-Me-DAB hepatocarcinogenesis: immunohistochemical and enzyme histochemical study

Immunohistochemical localization of gamma-glutamyl transpeptidase (gamma-GTP) in rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) hepatocarcinogenesis was investigated and compared with sites of gamma-GTP activity. Immunohistochemically, gamma-GTP was stained in the apical border of proliferating oval cells during the early stages of azo-dye carcinogen feeding. After 7 weeks, multiple hyperplastic nodules appeared in which gamma-GTP was localized in the bile canaliculi. In hepatoma tissues, positive staining for gamma-GTP was observed in the bile canaliculi-like spaces, on the cell membrane, and sometimes in the cytoplasm of malignant cells. Enzyme histochemical staining showed gamma-GTP activity to be present in almost the same areas as the immunoreactive gamma-GTP. However, some areas adjacent to hepatoma tissue showed immunohistochemically reactive protein but no enzyme activity. Immunoreactive gamma-GTP was present in all locations at which enzyme activity was seen. The present data suggest that an altered form of gamma-GTP might be present in tissues during 3'-Me-DAB hepatocarcinogenesis.