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PubMed1982

Detection of DNA damage in primary cultures of rat hepatocytes following in vivo and in vitro exposure to genotoxic agents.

A simplified method for the quantitation of DNA damage in nonlabeled hepatocytes, using a fluorometric technique for the quantitation of DNA in conjunction with a modification of the alkaline elution technique of Kohn et al [1976], following chemical treatment in vitro and in vivo, is described. Freshly isolated hepatocytes were treated in vitro with 2-acetylaminofluorene, aflatoxin B1, and dimethylnitrosamine, then examined for DNA damage. Exposure to each of these compounds resulted in DNA damage. Hepatocytes isolated from rats treated with the hepatocarcinogens 2-acetylaminofluorene, benzidine, azoxymethane, dimethylhydrazine, dimethylnitrosamine, and diethylnitrosamine sustained DNA damage as evidenced by increased alkaline elution. DNA damage in hepatocytes was also observed as a result of treatment with methylmethanesulfonate and azaserine. The hepatotoxin carbon tetrachloride did not induce DNA damage in hepatocytes isolated from treated animals. A comparison of the induction of DNA damage and of unscheduled DNA synthesis in hepatocytes from the same animals revealed that in most cases the extent of elution of DNA from filters was proportional to the induction of DNA repair.

Aflatoxin B1↗
PubMed1979

Enhancement of 2-acetylaminofluorene liver carcinogenesis in rats fed a choline-devoid diet.

The effects of feeding a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by 2-acetylaminofluorene (AAF) were investigated. Male Sprague-Dawley rats were fed either a CD or a CS diet containing 0.0075% AAF. Three to eight animals were killed 1, 3, 4 and 6 months thereafter. Well to moderately well differentiated hepatocellular carcinomas developed in 50% and 75% of rats fed the CD + AAF diet for 4 and 6 months, respectively. Cholangiocarcinomas developed, after 6 months, in 38% of the animals. No tumor developed in rats fed the CS + AAF diet. The results extend to AAF those previously obtained with DL-ethionine and azaserine showing that feeding a CD diet greatly potentiates the induction in rats of liver tumors by chemical carcinogens.

2-Acetylaminofluorene↗
PubMed1985

Regulation of purine biosynthesis in G1 phase-arrested mammalian cells.

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30-50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.

Animals↗
PubMed1990

Activation of c-Ki-ras not detectable in adenomas or adenocarcinomas arising in rat pancreas.

The objective of this study was to determine whether activation of c-Ki-ras occurs in carcinogen-induced rat pancreatic tumors. DNAs from 27 samples, which included adenomas, carcinomas in situ, and adenocarcinomas arising in azaserine-treated rats and corn oil-gavaged rats along with a nafenopin-induced rat pancreatic adenocarcinoma were examined for mutation of c-Ki-ras at codons 12, 13, and 61 by using the polymerase chain reaction. Our results indicate that activation of c-Ki-ras is not a common event during pancreatic carcinogenesis in the rat.

Adenocarcinoma↗
PubMed1993

Purine control of mouse oocyte maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest.

Hypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte-cumulus cell complexes. At a concentration of 1 mM, 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6-azauridine and 2,6-bis-(hydroxyamino)-9-beta-D-ribofuranosyl-purine had no effect. MPTF and 6-MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine-maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis-arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6-methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine-arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation.

Animals↗
PubMed1999

Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells.

Atherosclerotic vascular disease is a major complication of diabetic patients, and osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin, and azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (the key enzyme of the hexosamine pathway), profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Animals↗
PubMed1986

Accentuated response to soybean inhibitors by meal-feeding in various species.

Diets containing raw soybean products (RSD) fed ad libitum caused a reduction in food consumption and growth, pancreatic enlargement, hypersecretion of digestive enzymes, and enlargement of intestinal segments and their contents in rats, chicks and geese. These effects were found to be related to the concentration of trypsin inhibitor (TI) in the diet. Geese were more sensitive than rats and chicks. Calves fed RSD did not respond in pancreatic hypertrophy and hypersecretion of digestive enzymes, but growth was depressed. Meal-feeding also caused enlargement of the pancreas, digestive tract segments and their contents. The effects of feeding RSD and of meal feeding were found to be additive. In ad libitum feeding the animal can regulate food consumption and moderate the effects of antinutritional factors in the diet. Meal or tube-feeding interfered with this defense mechanism and the negative effects of RSD were accentuated and were lethal in non adapted rats and geese. In long-term studies the incidence of pancreatic nodules was correlated to the level of TI in the diet. Feeding RSD potentiated the carcinogenic effect of azaserine and meal feeding enhanced the incidence and size of the pancreatic nodules in rats fed RSD.

Amylases↗
PubMed1991

Residue trypsin inhibitor: data needs for risk assessment.

Trypsin inhibitor (TI) occurs naturally in many foods from plants, notably soybean protein products. Heat treatment inactivates TI and improves nutritional quality, but residual TI activity of 5 to 20% remains after typical commercial treatments. Chronic feeding of TI or products that contain TI can inhibit trypsin and chymotrypsin, stimulate their secretion, cause hypertrophy and hyperplasia of the pancreas, and lead to adenomas and carcinomas of the exocrine pancreas. In the rat, TI promotes pancreatic carcinogenesis initiated by azaserine. Data needed for possible risk assessment on TI would include 2-year bioassays from animals treated with TI and fed diets carefully controlled for type and amount of fat (which also promotes pancreatic carcinogenesis). The effects of TI on protein nutrition would have to be considered when identifying the maximum tolerated dose. Major reductions in human dietary TI exposure may not be feasible because of the multiple sources of TI, the substantial promotion by other factors such as fat, and the adverse effects of excessive heat on food products. For risk assessment of TI in a particular food, other promotors and the feasibility of decreasing TI intake must be considered.

Animals↗
PubMed1983

Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid.

Interspecific somatic cell hybrids were constructed between a Chinese hamster lung cell line deficient in hypoxanthine phosphoribosyltransferase and two lymphoblastoid cultures (GM 4025 and GM 3200) from unrelated males affected with the fragile (X) syndrome. Thirteen independent colonies survived selection in hypoxanthine-azaserine, while only one colony survived selection in hypoxanthine-aminopterin-thymidine. One hybrid formed from GM 4025 was found to contain a human X chromosome as the only detectable human chromosome in the majority of cells analyzed. Induction of fragile (X) expression in this hybrid at frequencies up to 20% was achieved by treatments with 5-fluoro-2'-deoxyuridine (5 X 10(-8) M or 1 X 10(-7) M) or methotrexate (5 X 10(-6) or 1 X 10(-5) for 12 h. Use of the somatic cell hybrid system may allow study of the fragile (X) from different patients on a homogeneous xenogeneic background and may provide a better system for characterization of the fragile (X) at the biochemical and molecular level.

Animals↗
PubMed1983

Methylamine metabolism and its role in nitrogenase "switch off" in Rhodopseudomonas capsulata.

In the photosynthetic bacterium Rhodopseudomonas capsulata, NH4+ switch-off of nitrogenase activity can be mimicked by its analog, methylamine. Like NH4+, methylamine appeared to require processing by glutamine synthetase (GS) before it was effective; gamma-glutamylmethylamide was shown to be the product of this reaction. Evidence that this glutamine analog functioned directly to initiate nitrogenase inactivation was suggested first by the fact that it was a poor substrate for glutamate synthase (i.e., it was not further metabolized by this pathway) and secondly, azaserine which blocks the transfer of the glutamine amide group had no effect on CH3NH3+ (or NH4+) switch-off. These observations are taken as preliminary evidence to suggest that when NH4+ inhibits nitrogenase activity, inactivation is initiated by glutamine itself, and not a molecule derived from it. Finally, evidence was presented that R. capsulata would use CH3NH3+ as a nitrogen substrate, but lag periods and generation times increased with subsequent passages.

Ammonia↗
PubMed1991

L-glutamine transport in native vesicles isolated from Ehrlich ascites tumor cell membranes.

Native vesicles isolated from Ehrlich ascites tumor cells accumulate glutamine by means of Na(+)-dependent transport systems; thiocyanate seems to be the more effective anion. The apparent affinity constant for the process was 0.38 mM. The Arrhenius plot gave an apparent activation energy of 12.3 kJ/mol. The structural analogs of glutamine, acivicin (2.5 mM) and azaserine (2.5 mM), inhibited the net uptake by 67 and 70%, respectively. The sulfhydryl reagents mersalyl, PCMBS, NEM, and DTNB also inhibited net uptake, suggesting that sulfhydryl groups may be involved in the activity of the carrier protein. A strong inhibition was detected when the vesicles were incubated in the presence of alanine, cysteine, or serine; in addition, histidine, but not glutamate or leucine, had a negative effect on glutamine transport.

Animals↗
PubMed1991

Transduction of the CHO aprt gene into mouse L cells using an adeno-5/APRT recombinant virus.

An adenovirus-5 recombinant virus Adapt1 carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene was constructed by insertion of a 2.5-kb fragment containing the complete CHO aprt structural gene linked to a Moloney murine sarcoma virus (MSV) promoter into the E3 region of adenovirus-5. The CHO aprt gene was in the opposite orientation to the adenovirus E3 promoter. Mouse Lapt- tk- (LAT) cells expressed the CHO aprt gene when infected with the virus, even at low MOI (O.1). APRT activity was detectable from approximately 20 h postinfection. At a low frequency, LAT cells were transformed to aprt+, and four stable transductants were selected in adenine, azaserine (AA) medium. Such cells expressed APRT at approximately 50% wild-type activity and the enzyme was shown to be CHO APRT by starch gel electrophoresis. DNA was isolated from the transductants and probed with CHO aprt-specific DNA and with viral DNA probes. The results indicated that the CHO aprt gene was integrated into the LAT cells at a site other than mouse aprt. Although neighboring viral sequences were integrated and maintained in the transductants, viral sequences further upstream and downstream of the aprt gene were absent.

Adenine↗
PubMed1985

Dependence of nitrate utilization upon active CO2 fixation in Anacystis nidulans: a regulatory aspect of the interaction between photosynthetic carbon and nitrogen metabolism.

Specific inhibition of photosynthetic CO2 fixation in Anacystis nidulans cells by D,L-glyceraldehyde resulted in the simultaneous inhibition of nitrate utilization, indicating a dependence of the latter process upon the provision of CO2-fixation products. This dependence was lost in cells treated with L-methionine-D,L-sulfoximine or azaserine, effective inhibitors of ammonium assimilation. In these cells, nitrate uptake could proceed at rates similar to those in control cells even if CO2 fixation was severely inhibited by D,L-glyceraldehyde. The results support the contention that CO2-fixation products participate in the control of nitrate uptake in A. nidulans by preventing the accumulation of certain ammonium derivatives which are negative effectors of nitrate uptake.

Ammonia↗
PubMed1992

Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves.

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). D-Isomers of alanine and isoleucine were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the alpha-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that beta-alanine and analogues with methylation at the alpha-carbon, at the carboxyl group, or at the alpha-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the alpha-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the beta-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.

Alanine↗
PubMed1984

Mode of action of 2-amino-6-chloro-1-deazapurine.

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5'-phosphate which accumulated to concentrations as high as 1000 nmoles/10(9) cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibit the synthesis of 5'-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Aminopurine↗
PubMed1989

Inhibition of CCRF-CEM human leukemic lymphoblasts by triciribine (tricyclic nucleoside, TCN, NSC-154020). Accumulation of drug in cells and comparison of effects on viability, protein synthesis and purine synthesis.

The experimental antineoplastic agent triciribine (tricyclic nucleoside, TCN) is known to be activated to its phosphate TCN-P by adenosine kinase and to inhibit cell growth, purine nucleotide synthesis, and incorporation of amino acids into proteins. Our objective in this paper was to compare these effects in intact cells of a human cell line as a prerequisite to describing in a companion paper [Moore et al., Biochem. Pharmac. 38, 4045 (1989)] more detailed enzymic studies of their interrelationships. TCN treatment inhibited cloning of CCRF-CEM human leukemic lymphoblasts 50% at concentrations of 6, 30, and 90 microM with 8-day, 8-hr, and 2-hr exposures respectively. However, 6-20% of the cells survived exposure to 200 microM TCN for 24 hr. The intracellular formation of TCN-P from TCN was rapid, concentrative and essentially complete, but TCN-P did not exceed about 1.4 mM (1.4 nmol/10(6) cells) at 200 microM TCN. In cells exposed to 50 microM TCN for 1.25 to 24 hr, formate incorporation into ATP and GTP was inhibited the most rapidly and strongly; pools of ATP and GTP were decreased as much as 40% (as compared with controls); and incorporation of formate into RNA purines was inhibited as much as 65%. Incorporation of leucine into protein was more moderately inhibited up to 40%, apparently in proportion to the concentration of intracellular TCN-P, rather than of the TCN in the medium. These inhibitions occurred most rapidly during the first 2-4 hr and increased only gradually thereafter, whereas cloning ability was inhibited more slowly and uniformly over a longer time period. No one of these metabolic effects by itself showed a clear correlation with the loss of viability. The incorporation of formate into formylglycinamide ribotide (FGAR, when accumulated at a blockage by azaserine) was inhibited drastically by TCN. The rate of incorporation of hypoxanthine into ATP was increased by TCN, whereas incorporation into GTP was decreased. Thus, the principal sites of inhibition of purine synthesis by TCN-P were shown in these intact cells to be at a step prior to synthesis of FGAR in the de novo pathway and also at an additional site between IMP and GTP.

Adenosine Triphosphate↗
PubMed1984

Human GMP synthetase.

The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes, placenta, and liver. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration. The Km values were determined to be 4.9 microM for XMP, 270 microM for ATP, and 340 microM for glutamine. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient. The enzyme was specific for ATP as the energy source. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

Carbon-Nitrogen Ligases↗
PubMed1996

Effects of bradykinin on the intracellular calcium concentration of pancreatic acinar AR42J cells.

We examined the effects of bradykinin (BK) on the intracellular free calcium concentration ([Ca2+]i) in rat pancreatic acinar AR42J cells. BK induced a dose-dependent rise in [Ca+2]i in AR42J cells between the concentrations of 10(-12)M and 10(-7)M. The BK-evoked response was not affected by the presence of Co2+ or the absence of extracellular calcium. This response was suppressed by neomycin or the B2 antagonist, but not by the B1 antagonist. The response was also attenuated by treatment with dexamethasone. These results suggest that BK increases [Ca2+]i through the B2 receptors by promoting the phosphatidyl inositol turn-over and that, in the process of azaserine-induced undifferentiation, the pancreatic acinar cells strongly express the BK receptors.

Animals↗