Mucosal polyamine measurements and colorectal cancer risk

Polyamines are short-chain aliphatic amines required for normal cellular growth that are ubiquitously found in all living tissues. Polyamine content has been shown to correlate with cellular proliferation. Quantitation of polyamines may thus provide a biochemical measure of proliferation in the colorectal mucosa where dysregulated epithelial proliferation is associated with colorectal cancer risk. A case-control study was conducted to validate the hypothesized association between mucosal polyamine measurements and colorectal cancer risk. Polyamines were measured in 4–6 multiple rectal mucosal biopsies from 11 normal control subjects and seven case patients with colon cancer. Compared with the controls, mean polyamine measurements, after adjustment for age and sex, were significantly increased for spermidine (P < 0.003) and spermine (P < 0.017). Subsequent analyses indicated that in controls 1–4 biopsies appeared adequate to characterize an individual. However, mucosal polyamines in the cases exhibited more sampling variability, requiring 4–8 biopsies to achieve an acceptable level of reliability. After adjustment for age and sex, the odds ratios for spermidine and spermine levels, compared to the controls, were 4.8 (95% confidence interval: 1.6–33.7) and 2.3 (1.2–6.3), respectively. The results of this study indicate that increases of mucosal polyamine measurements, after taking the sampling and methodological variability into account, are significantly associated with colorectal cancer risk, and suggest that polyamine measurements in rectal mucosa may play an important role as biomarkers for identifying high-risk individuals and/or for using as intermediate endpoints in prevention trials. © 1996 Wiley-Liss, Inc.     

Polyamine Levels During Growth, Sporulation, and Spore Germination of Bacillus megaterium

Spermidine was the major (>95%) polyamine of Bacillus megaterium in all stages of growth, although it could be replaced completely by spermine. Log-phase cells had 40 to 50% as much spermidine, based on ribonucleic acid (RNA) content, as did either stationary-phase cells or dormant spores; similar results were obtained in three other bacilli including an asporogenous mutant. Polyamine levels were essentially the same in B. megaterium grown in rich or poor media, or in media of high or low ionic strength. Polyamine levels were elevated three- to sixfold by exogenous spermidine without a major effect on growth, sporulation, or subsequent spore germination. During germination, the absolute amount of spermidine remained constant for almost 2 h until net RNA synthesis had lowered the polyamine/RNA ratio to a value close to that in log-phase cells. At this time, the spermidine level began to rise, and thereafter spermidine and RNA increased in parallel. This parallel relationship between the spermidine and RNA levels was abolished by actinomycin D, but not by chloramphenicol.     

Comparison of seasonal growth and polyamine content in shoots of orchard-grown standard and genetic dwarf apple tress

Seasonal variations in growth rate, and polyamine and arginine content were determined in shoots of standard and genetic dwarf apples ( Malus domestica Borkh.) in orchard conditions. Putrescine, spermidine, spermine and arginine exhibited the same seasonal variations. Polyamine titer and arginine content were correlated. Putrescine, spermidine, spermine and arginine levels increased during March and April, reaching the maximum at the end of April. From the onset of May, they decreased gradually until they reached a steady level at the end of July and beginning August. Then the level increased until September when growth stopped. Shoots of both standard and dwarf trees started to grow in April, elongated slowly during the summer and ceased growth in September. Polyamine and arginine content of the shoots of standard apple trees were higher compared to the shoots of genetic dwarfs, but the concentration of polyamine and arginine were not correlated with the growth rate.     

Comparison of soybean cultivars for enhancement of the polyamine contents in the fermented soybean natto using Bacillus subtilis (natto)

Polyamines have beneficial properties to prevent aging-associated diseases. Raw soybean has relatively high polyamine contents; and the fermented soybean natto is a good source of polyamines. However, detailed information of diversity of polyamine content in raw soybean is lacking. The objectives of this study were to evaluate differences of polyamines among raw soybeans and select the high polyamine-containing cultivar for natto production. Polyamine contents were measured chromatographically in 16 samples of soybean, which showed high variation among soybeans as follows: 93–861 nmol/g putrescine, 1055–2306 nmol/g spermidine, and 177–578 nmol/g spermine. We then confirmed the high correlations of polyamine contents between raw soybean and natto (r = 0.96, 0.95, and 0.94 for putrescine, spermidine, and spermine, respectively). Furthermore, comparison of the polyamine contents among 9 Japanese cultivars showed that ‘Nakasen-nari’ has the highest polyamine contents, suggesting its suitability for enhancement of polyamine contents of natto.     

Epidermal keratinocytes actively maintain their intracellular polyamine levels

Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation. However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed. The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels. The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations. At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine. Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine. Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium. At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium. We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium. Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function.     

THE UPTAKE OF SPERMIDINE AND SPERMINE BY SLICES OF MOUSE CEREBRAL HEMISPHERES

Abstract— Spermidine and spermine are taken up into mouse cerebral hemisphere slices by active transport and can be accumulated well above the medium concentration. The uptake process shows saturation kinetics and resembles that for amino acid uptake in that it is sensitive to temperature and inhibited by cyanide, 2,4-dinitrophenol or by the absence of glucose from the medium. However, at low initial medium concentrations spermine is taken up by a process which is insensitive to metabolic inhibitors or to temperature. It is suggested that either physical binding to a cellular constituent or exchange transport may account for this uptake. Ouabain does not inhibit polyamine uptake. Spermidine or spermine uptake is inhibited by cadaverine and putrescine. Spermine is the most potent inhibitor of spermidine uptake and vice-versa. Polyamine uptake differs from that of amino acids in that it is increased by a reduction in medium sodium or calcium content and decreased by an increase in medium potassium content. Recently taken up spermine undergoes heteroexchange with spermidine and homoexchange with recently entered spermine. Spermidine undergoes neither heteroexchange with spermine nor homoexchange.     

Effect of repeated severe pruning on endogenous polyamine content in hazelnut trees

Endogenous polyamine content was determined in leaves and buds of adult and repeatedly severe pruned hazelnut trees ( Corvlus avellana L.). Polyamine content in leaves from shoots obtained by forced outgrowth of branches taken from adult and pruned trees was also determined. Variations of polyamine levels in relation to pruning treatments were observed in all the analysed, tissues. Free polyamines increased in response to pruning treatments, mostly due to an increase in free putrescine. Free spermidine and spermine seemed to decrease with pruning intensity, whereas bound polyamines did not seem to correlate with treatments. Significantly, in all the analysed tissues the putrescine to spermidine plus spermine ratio increased in the free polyamine fraction. The results indicate that polyamine metabolism could play a role as a physiological marker for juvenility and rejuvenation in relation to cloning of woody plants. The possible role of polyamines in mediating and/or regulating phase change and reinvigoration is discussed.     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.     

Proliferative potential and expression of cell type specific functions in primary mouse colonic epithelial cells

Summary Primary cultures of mouse colonic epithelial cells have been obtained that are typically epithelial by morphology and moreover express keratins and endogenousβ-galactosidase; this latter activity was also demonstrated in the epithelial lining of the mouse colonic mucosa. The proliferative response of the primary colonic epithelial cells to epidermal growth factor, insulin, and the bile acid, deoxycholic acid, has been studied. Using primary cultures maintained at suboptimal growth conditions, which yielded 96 to 100% quiescent cells, epidermal growth factor, insulin, and the bile acid, deoxycholic acid, at concentrations at which it normally occurs in the aqueous phase of human feces, stimulated proliferation as measured by autoradiography. Exposure of the cells to combinations of these factors resulted in additive increases in growth. In conclusion, cells from the normal mouse colon can now be cultured while retaining at least two normal marker functions and moreover respond to some known mitogens and the potential tumor promoter deoxycholic acid. The cells can also be subcultivated while maintaining their epithelial morphology and marker functions for at least 3 passages.     

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.     

Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter.

We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent Km values of 0.13 mM for spermine, 0.26 mM for spermidine, and 1 mM for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

Histamine prevents polyamine accumulation in mouse C57.1 mast cell cultures.

The effects of histamine on polyamine uptake and metabolism was studied in a mouse mast cell line (C57.1), as a cell model in which both biogenic amines are important for maintaining cell function and viability. Results obtained after incubations with exogenous histamine indicated that histamine prevents polyamine accumulation by affecting polyamine uptake. A plasma membrane transport system for polyamines has been also studied in mast cells. It seems to be a Na(+)-dependent uptake with high affinity for both spermine and spermidine and lower affinity for putrescine and agmatine. Polyamine uptake was reduced in both cells treated with exogenous histamine and histamine-preloaded cells. However, ornithine decarboxylase activity and cell proliferation were not affected by histamine. Incubation with histamine enhanced the spermidine/spermine acetyl transferase induction caused by N(1)-ethyl-N(11)-[(cyclopropyl)methyl]-4,8-diazaundecane, suggesting that polyamine acetylation could be another mechanism by which histamine prevents polyamine accumulation in C57.1 mast cells.     

Effects of Exogenous Manganese (Mn) on Mineral Elements,Polyamines and Antioxidants in Apple Rootstock <i>Malus robusta</i> Rehd.

Manganese (Mn) is one of the essential microelements in all organisms. However, high level of Mn is deleterious to plants. In this study, the effects of exogenous manganese application on mineral element, polyamine (PA) and antioxidant accumulation, as well as polyamine metabolic and antioxidant enzyme activities, were investigated in Malus robusta Rehd., a widely grown apple rootstock. High level of Mn treatments decreased endogenous Mg, Na, K and Ca contents, but increased Zn content, in a Mn-concentration-dependent manner. Polyamine metabolic assays revealed that, except the content of perchloric acid insoluble bound (PIS-bound) spermine, which increased significantly, the contents of putrescine (Put), spermidine (Spd) and spermine (Spm) all decreased progressively, accompanied with the decreased activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), and the increased activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.5.3.3). Further antioxidant capacity analyses demonstrated that contents of anthocyanin, non-protein thiols (NPT) and soluble sugar, and the activities of guaiacol peroxidase (POD, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1), also increased upon different concentrations of Mn treatments. Our results suggest that endogenous ion homeostasis is affected by high level of Mn application, and polyamine and antioxidant metabolism is involved in the responses of M. robusta Rehd. plants to high level of Mn stress.     

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.     

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages

To further evaluatethe role of polyamines in insulin production and cell replication indiabetic pancreatic islets, we have studied hyperplastic islets ofobese-hyperglycemic mice of different ages and normal islets of thesame strain. The aims of the study were to investigate the impact ofthe diabetic state and aging on polyamine contents and requirements inthese islets. Cultured islets from lean and obese animals containedsignificantly less polyamines than freshly isolated islets.Spermine-to-spermidine ratio was elevated in freshly isolated isletsfrom young obese mice compared with those from lean mice. In isletsfrom old obese animals, spermidine content was decreased, whereas thecontent of spermine was not different from that of young obese mice.The physiological significance of polyamines was investigated byexposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets butfailed to affect polyamine content of cell nuclei. Insulin content wasnot affected in polyamine-deficient islets of obese mice, irrespectiveof age, in contrast to decreased islet insulin content inpolyamine-depleted young lean animals. Polyamine depletion depressedDNA synthesis rate in obese mouse islets; in lean mice it actuallystimulated DNA synthesis. We concluded that important qualitative andquantitative differences exist between islets from obese-hyperglycemicand normal mice with respect to polyamine content and requirements ofpolyamines for regulation of insulin content and cell proliferation.The results suggest that spermine may be involved in mediating therapid islet cell proliferation noted early in obese-hyperglycemicsyndrome, but changes in spermine concentration do not seem to accountfor the decline in islet cell DNA synthesis in aged normoglycemic animals.

     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Oxidation of Polyamines by Fungal Enzymes

Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen. The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate. The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H₂O₂. Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.     

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.