Decreased vacuolar acidification capacity in drug-resistant rat liver preneoplastic nodules.

Rat liver nodules produced by intermittent 2-acetylaminofluorene feeding exhibit alterations in cell surface receptors reminiscent of impairment of vacuolar acidification. In this report, vacuolar acidification activity, measured as the ATP-dependent quenching of acridine orange, was characterized in liver and nodular membrane fractions using various ion-transport inhibitors and with respect to nucleotide specificity and divalent cation dependence. Based on these criteria and on the comparison of vacuolar acidification activity with mitochondrial, lysosomal, and plasma membrane marker enzymes in different subcellular fractions, it was concluded that the assay measures the proton pump associated with exocytic and/or endocytic vacuolar compartments. When the vacuolar acidification activity was compared in liver and nodular subcellular membrane fractions, it was found that the vacuolar acidification was most strongly reduced in nodular low-density membrane fractions enriched in Golgi-derived membranes and endocytic vesicles. The data indicate that vacuolar, i.e., exocytic and/or endocytic, prelysosomal intracellular compartments in rat liver nodules are markedly deficient in acidification capacity, possibly providing an explanation to various metabolic aberrations, such as diminished iron accumulation and reduced protein degradation, observed in rat liver nodular cells.

Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats.

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.

Lipid compositions of intracellular membranes isolated from rat liver nodules in Wistar rats.

The mevalonate pathway gives rise to important end products for the regulation of growth and resistance to oxidative stress and is, consequently, of importance in carcinogenesis. In this study liver nodules were produced in Wistar rats by intermittent feeding with dietary 2-acetylaminofluorene, and the lipid compositions of isolated microsomes, mitochondria, and lysosomes were examined. The phospholipid compositions of these subfractions were unchanged compared to normal hepatic tissue, but the fatty acid patterns were altered, particularly in microsomes. An increase in the content of palmitic acid and a decrease in that of stearic acid were noted. The pattern of fatty acyl moieties on carbon atoms 1 and 2 of the glycerol backbone of phospholipids was unchanged in nodular tissue compared to normal liver. The amount of dolichol was significantly higher in microsomes and mitochondria, but not in lysosomes, and the relative amounts of longer polyisoprenoid compounds were increased in the liver nodules. The relative concentration of esterified dolichol was decreased and an enrichment in saturated fatty acids in this fraction could be observed. The cholesterol concentration was found to be lower in microsomes, but was unchanged in mitochondria and lysosomes, and the normally low concentration of cholesteryl esters was elevated somewhat in microsomes and lysosomes. The ubiquinone content of liver nodular mitochondria was unchanged, but increased 7-fold in microsomes and 2-fold in lysosomes. The alterations found in the lipid composition of liver nodules are significant and have functional implications in many cellular processes of proposed importance for the carcinogenic process, i.e., protein glycosylation cholesterogenesis, regulation of the mevalonate pathway, cellular oxidation-reduction state, and resistance to oxidative stress.

Role of dolichyl phosphate in regulation of protein glycosylation in 2-acetylaminofluorene-induced carcinogenesis in rat liver.

Hyperplastic nodules and hepatocarcinomas were produced in rat liver by 2-acetylaminofluorene-containing diet. The homogenates and isolated microsomes were analyzed for the content of lipid intermediates and glycosylation reactions. The dolichol content of hyperplastic nodules increases four times in the homogenate and six times in the microsomes. In developed hepatocarcinoma, the amount of dolichol was doubled. Concerning the distribution pattern of the polyprenols, there is a change in the relative amounts of dolichols with 18 and 19 residues. In contrast to the free alcohol, dolichyl phosphate was greatly decreased in nodules, a finding which might be explained by a decreased dolichol kinase and an increased dolichol monophosphatase activity. The percentage of total phosphorylated dolichol was related to the glycosylating capacity. In microsomes, mitochondria, and homogenate from normal liver and in homogenate from hyperplastic liver nodules, the percentages of dolichyl phosphate were 23, 2, 16, and 4, respectively. At maximal glycosylation in vitro, only part of the total dolichyl phosphate was glycosylated. Dolichol-mediated protein glycosylation exhibited a general decrease in the microsomes from nodules and cancer tissue; it is suggested that the main cause of the decrease is a shortage of the available dolichyl phosphate which is rate limiting and which also contributes to the synthesis of the modified oligosaccharide chain.

Increased uridine diphosphate-glucuronyltransferase activity in preneoplastic liver nodules and Morris hepatomas.

In preneoplastic rat liver nodules produced by 2-acetylaminofluorene, certain uridine diphosphate-glucuronyltransferase (UDP-GT) activities, which are ascribed to a distinct enzyme form, were selectively increased (5-fold). This enzyme form, operationally termed UDP-GT1, accepts 1-naphthol,4-methylumbelliferone, and 3-hydroxybenzo(a)pyrene as substrates and is chiefly inducible in liver by 3-methylcholanthrene-type inducers. Glucuronidation of other substrates (morphine, 4-hydroxybiphenyl, chloramphenicol, bilirubin, and estrone) was only slightly enhanced or decreased in nodular tissue. Differentially increased UDP-GT1 activities were also found in Morris hepatomas 9121 and 7777. Rabbit antibodies to rat liver UDP-GT1, purified from 3-methylcholanthrene-treated rats, demonstrated immunological similarity between the enzymes from liver, nodular tissue, and Morris hepatoma 9121. Rocket immunoelectrophoresis ascertained that enhanced enzyme activity in nodular tissue reflected an increased level of enzyme protein. Increased activity of UDP-GT1 together with decreased cytochrome P-450-dependent monooxygenase may contribute to the resistance of preneoplastic hepatocytes to the cytotoxic actions of chemical carcinogens.

Expression of glutathione transferase pi as a predictor for treatment results at different stages of acute nonlymphoblastic leukemia.

The expression of glutathione transferase pi (GST pi) was studied in leukemic cells from 60 patients with acute nonlymphoblastic leukemia at diagnosis and at progressing stages of the disease. A polyclonal rabbit antibody to human placental GST pi coupled with peroxidase antiperoxidase staining was used for immunodetection of GST pi on sections of routinely fixed bone marrow clots. All patients had received induction therapy based on an anthracycline and a standard dose of ara-C. The expression of GST pi at diagnosis was significantly correlated with response to induction therapy, duration of first remission, and overall survival. Twenty-nine of 36 samples of bone marrow from patients that entered complete remission (CR) following primary induction therapy showed a low expression, whereas nine of 16 sections from patients with resistant disease showed a high expression of GST pi (P less than or equal to 0.03). Of 40 sections that showed a low expression of GST pi, 29 (73%) were taken from patients that achieved a CR, whereas 12 of 19 sections that showed a high expression of the enzyme were from patients with resistant disease or that entered CR only after additional therapy (P less than or equal to 0.02). The median duration of first CR was 18.2 mo for patients whose cells showed a low expression of GST pi compared with 6.7 mo for those that entered CR in spite of a high expression of the enzyme (P less than or equal to 0.005). Of cells from ten patients that at the time of study were in a continuous first CR, none expressed high concentrations of GST pi. The expression of GST pi remained rather constant in most patients as the disease progressed to clinical resistance. At relapse there was no significant correlation between the expression of GST pi and treatment results but, of ten patients that entered a second CR or achieved a partial remission, only one showed a high expression of the enzyme. We conclude that there was a significant correlation between the expression of GST pi at the time of diagnosis and the subsequent treatment results and that GST pi is a useful marker for clinical resistance to cytostatic drugs in acute nonlymphoblastic leukemia.

Persistently increased expression of a 3-methylcholanthrene-inducible phenol uridine diphosphate-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas.

Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas produced by feeding 2-acetylaminofluorene or N-nitrosomorpholine was studied using isozyme-selective substrates, antibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) activities toward 4-methylumbelliferone, 1-naphthol, and benzo[a]pyrene-3,6-quinol were reversibly increased by short term feeding of 2-acetylaminofluorene but were persistently increased in hepatocyte nodules and differentiated hepatocellular carcinomas. Immunoblot analysis revealed that short term feeding of 2-acetylaminofluorene increased a Mr 55,000 polypeptide corresponding to the previously characterized UDP-GTI or phenol UDP-GT. However, in some hepatocyte nodules and hepatocellular carcinomas either the Mr 55,000 or a new Mr 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte nodules, which may contribute to the toxin-resistance phenotype frequently observed at cancer prestages.

Preparation of liver microsomes with high recovery of endoplasmic reticulum and a low grade of contamination.

A modified procedure for preparing the microsomal fraction from rat liver was developed with the aim of increasing the recovery without increasing the degree of contamination. 87% of the membranes of the microsomal fraction isolated from the first mitochondrial (10 000 X g) supernatant originates from the endoplasmic reticulum, representing a 35% yield. By gentle resuspension of the 10 000 X g pellet followed by differential centrifugation a second crop of microsomes can be prepared which, together with the first crop, gives a 55% total recovery of microsomal markers. 87% of the protein in this second crop also originates from the endoplasmic reticulum and this fraction has properties similar to those of the first crop. Contaminating membranes include Golgi membranes (0.6% of the total protein), mitochondria (2.5%), lysosomes (5%) and plasma membranes (5%). Collecting further crops increases the contamination. Subfractionation studies revealed almost identical distributions of ribosome-rich, ribosome-poor and smooth membranes in the two crops of microsomal fractions. The results obtained after treatment of the animals with phenobarbital or methylcholantrene were similar to those obtained with control animals; but in the case of methylcholantrene treatment the second crop represents a larger portion of the total membranes of the endoplasmic reticulum.

Characterization of UDP-galactosyl:asialo-mucin transferase activity in the Golgi system of rat liver.

UDPgalactosyltransferase activity (UDPgalactose:mucopolysaccharide galactosyltransferase, EC 2.4.1.74) was measured in a well-characterized fraction of Golgi membranes in the presence of UDPgalactose and exogenous acceptor sites. Substrate saturation for 0.05 mg Golgi protein was achieved at a concentration of 4.6 mM UDPgalactose. Desialylated mucin proved to be the most suitable acceptor protein. Access to galactose acceptor sites was not rate limiting for the reaction when 20 mg of asialo-mucin/ml of incubation mixture was used. With these concentrations of substrates the use of nucleotides to inhibit pyrophosphatases and of detergents to perturb the membrane structure was not necessary and proved, in fact, to be inhibitory to galactose transfer. UDPgalactosyl:asialo-mucin transferase activity in Golgi membranes was 230 nmol galactose transferred/mg Golgi protein per 30 min.

Studies on the latency of UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal and Golgi subfractions from rat liver.

The activity of UDPgalactose-asialo-mucin galactosyltransferase (EC 2.4.1.74) in microsomal and Golig subfractions was stimulated 2.4-fold after disruption of the membrane permeability barrier by hypotonic incubation. In the presence of Triton X-100, galactose transfer to asialo-mucin was increased 12-fold in rough microsomes and 5-fold in smooth microsomes both with and without hypotonic incubation; while in the Golgi subfractions no stimulation by detergent was observed. These experiments indicate differences in enzyme-lipid or enzyme-protein interactions in microsomes and Golgi membranes. Furthermore, these results strongly support the conclusion that the UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal fractions is not due to contamination by Golgi vesicles but represents an enzyme activity endogenous to the endoplasmic reticulum.

Sequential preparation of rat liver microsomal and Golgi membranes.

A new procedure for the preparation of microsomes, microsomal subfractions and Golgi membranes from the same piece of rat liver has been developed. The smallest amount of liver with which the preparation can be performed is about 1 g (wet weight). 35% of the total activity of marker enzymes for the endoplasmic reticulum was recovered in the microsomal fraction. This recovery is approximately the same as that obtained in our laboratories using other procedures. Golgi membranes, mitochondria, lysosomes and plasma membranes represent less than 13% of the microsomal protein as calculated on the basis of marker enzymes. Golgi membranes must be prepared in two steps to achieve a reasonable recovery and thus a representative sample containing both very low density lipoprotein-rich Golgi vesicles and the heavier cisternal elements. The recovery of UDP-galactosyltransferase activity in the Golgi fraction from the livers of alcohol-treated animals is around 30% of the total activity in the total particulate fraction.

The topology of epoxide hydratase and benzpyrene monooxygenase in the endoplasmic reticulum of rat liver.

The distributions of benzpyrene monooxygenase and epoxide hydratase in subfractions of liver microsomes from control and from phenobarbital- and methylcholanthrene-treated rats have been investigated. The specific activities of these enzymes in rough and smooth microsomes from control and phenobarbital-treated animals are approximately the same, whereas after methylcholanthrene treatment benzpyrene monooxygenase is four times higher and epoxide hydratase twice as high in the rough vesicles. Further subfractionation of rough and smooth microsomes by rate differential centrifugation revealed the distributions of both enzymes among microsomal vesicles to be highly heterogeneous. Comparison of these distributions leads to the conclusion that the benzpyrene monooxygenase system and epoxide hydratase may form a complex of unique stoichiometry in the membrane of microsomes from control rats, but that such a complex is not consistent with the distributions obtained after methylcholanthrene induction. Studies with proteases and the nonpenetrating chemical reagent diazobenzene sulfonate suggest that epoxide hydratase may be buried deeply in the hydrophobic phase of the membrane of the hepatic endoplasmic reticulum.

NADH oxidase of liver plasma membrane stimulated by diferric transferrin and neoplastic transformation induced by the carcinogen 2-acetylaminofluorene.

NADH oxidase of purified plasma membranes (electron transfer from NADH to oxygen) was stimulated by the growth factor diferric transferrin. This stimulation was of an activity not inhibited by cyanide and was not seen in plasma membranes prepared from hyperplastic nodules from liver of animals fed the hepatocarcinogen, 2-acetylaminofluorene, nor was it due to reduction of iron associated with diferric transferrin. With plasma membranes from nodules, the activity was already elevated and the added transferrin was without effect. The stimulation by diferric transferrin did not correlate with the absence of transferrin receptors which were increased at the nodule plasma membranes. With liver plasma membranes, the stimulation by diferric transferrin raised the plasma membrane NADH oxidase specific activity to approximately that of the nodule plasma membranes. In contrast to NADH oxidase, which was markedly stimulated by the diferric transferrin, NADH ferricyanide oxidoreductase or reduction of ferric ammonium citrate by liver plasma membranes was approximately equal to or slightly greater than that of the nodule plasma membrane and unaffected by diferric transferrin. The results suggest the possibility of coupling of NADH oxidase activity to a growth factor response in mammalian cells as observed previously for this enzyme in another system.

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat.

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.

Selective expression of glutathione transferase isoenzymes in chemically induced preneoplastic rat hepatocyte nodules.

Isoenzymes of glutathione transferase were shown to occur at selectively altered levels in rat hepatocyte nodules produced by 2-acetylaminofluorene treatment. Changes were measured by different substrates, antibodies raised against purified glutathione transferases, and by purification of the major isoenzymes. Isoenzymes composed of subunits 1, 2 and 3, expressed in normal liver tissue, all occurred at increased concentrations in nodules, whereas the level of transferase 4-4 was decreased. The most conspicuous change was the appearance of glutathione transferase 7-7 (or transferase P), the concentration of which in negligible in normal liver.

Ubiquinone is reduced by lipoamide dehydrogenase and this reaction is potently stimulated by zinc.

Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 microM in the presence and 4 microM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.

Bone marrow cells in patients with multiple sclerosis.

Bone marrow cells from patients with multiple sclerosis (MS) were studied regarding proliferative capacity with and without mitogenic stimulation, immunohistochemical characterization of cellular phenotypes with monoclonal antibodies and morphology and compared to bone marrow cells from healthy individuals undergoing elective orthopaedic surgery. MS patients' bone marrow mononuclear cells (BM-MNC) showed higher spontaneous proliferation both in comparison with BM-MNC from controls and peripheral blood mononuclear cells (PBL) from MS patients. Phytohaemagglutinin (PHA) response was higher in MS BM-MNC than BM-MNC from controls. MS patients' BM-MNC proliferated more on interleukin-2 (IL-2) stimulation than their corresponding PBL. There was no significant difference in proliferative response of PBL between MS patients and controls. Higher levels of undifferentiated or activated cells, as measured by OKT10, were found in peripheral blood of patients with MS. Seven of 11 MS patients showed morphological signs of activation in their bone marrow. The results indicate a role for immune reactions in bone marrow in the pathogenesis of multiple sclerosis, a disease with symptoms and signs strictly confined to the central nervous system.

Hepatocellular cancer development in spleen of syngenic rats after transfer of potential precursor hepatocytes.

An in vitro-in vivo approach was used to examine the effect of selected cell proliferation on the progression to cancer. Liver cells isolated from rat donors with nodular livers were kept for 3 h in vitro, transferred to spleens and livers of syngeneic recipients and assessed at 4 and 10 months for evidence of cancer. Ten months after transfer of one million nodular liver cells (40% were GST-P + hepatocytes) to each of 50 recipients, 13 rats developed 21 hepatocellular cancers in their spleens (11 cancers) and livers. The latency to cancer was shortened by exposure to 2-acetylaminofluorene of the recipient rats at the time of hepatocyte transfer.

Growth hormone administration after treatment in the resistant hepatocyte model does not affect progression of rat liver carcinogenesis.

Male and female Wistar rats were treated according to a slightly modified resistant hepatocyte model, i.e. initiation with diethylnitrosamine and selection of initiated cells with 2-acetylaminofluorene and partial hepatectomy. Two weeks after selection, rats of each sex received daily subcutaneous injections of either recombinant human growth hormone (2.5 IU/kg) or saline for 6 weeks. No effects on growth of early enzyme-altered liver lesions were recorded. The long-term part of the experiment did not show any differences due to growth hormone treatment in terms of incidence or latency time for development of either malignant liver tumors or kidney tumors. Male rats developed liver tumors more frequently than the female rats whereas a higher incidence of kidney tumors was observed in the female rats. Several different malignancies at other sites were also recorded, with no differences between the groups with or without growth hormone treatment. In conclusion, no modifying effects of human growth hormone administration during the post selection phase of the resistant hepatocyte model could be demonstrated on either tumor promotion or tumor progression.

Nitrated polycyclic aromatic hydrocarbons: a risk assessment for the urban citizen.

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are formed during incomplete combustion. Sources include emissions from vehicles (mainly diesel vehicles), heating, smoking, certain types of food processing, and incomplete combustion in general. Nitro-PAHs are direct-acting mutagens, and a number of them have been shown to be carcinogens. 2-Nitrofluorene (NF) represents a model substance for the nitro-PAHs. An attempt has been made to calculate the human cancer risk due to exposure to nitro-PAHs by two different models. In the first model, genotoxic lesions were transferred to units of Gray (gamma-irradiation), and in the second model a mega study (24,000 animals) on the carcinogenicity of one metabolite of NF was used to elucidate the risk. Gamma-irradiation of the rat liver gave rise to preneoplastic foci in a dose-dependent manner, which was statistically significant. The Gray-equivalents of chemically (NF) induced foci were calculated, and from the human nitro-PAH exposure, expressed in Sievert, a human risk estimate was calculated. In the second model, an extrapolation from laboratory animals to man was performed because tumor data on 2-acetylaminofluorene (AAF), a major metabolite of NF, were available in the literature. The tumor dose-response data on AAF was linear for tested lifetime doses. The results of both models agreed, with a risk range of 0.15-49 x 10(-6) on human cancer risk for an urban citizen.