1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels in rat distal colonic mucosa: effects of 2-difluoromethylornithine

Recently, our laboratory has demonstrated that N1-acetylspermidine levels were increased in the distal colonic mucosa of rats administered 1,2-dimethylhydrazine for 15 and 26 weeks. In order to further explore the possible role of this acetylated polyamine in the malignant transformation process induced by this carcinogen, groups of rats were subcutaneously injected weekly with dimethylhydrazine (20 mg/kg body wt.) or diluent for 5, 10, 15 and 26 weeks +/- 1% 2-difluoromethylornithine in the drinking water. The latter agent, an irreversible inhibitor of ornithine decarboxylase, has previously been shown to inhibit colonic tumor formation in this experimental model. At each of these time periods, rats from each group were killed, their proximal and distal colonic mucosa harvested and examined, and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase. The results of these experiments demonstrated that: (1) N1-acetylspermidine levels in the proximal colonic segment of all animals were similar at each time point; (2) N1-acetylspermidine levels were also similar in the distal colons of all animals at 5 and 10 weeks. At 15 weeks, however, the level of N1-acetylspermidine was increased in the dimethylhydrazine-treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (3) at 26 weeks, the level of this acetylated polyamine remained higher in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was markedly elevated in colonic tumors; (4) co-administration of difluoromethylornithine decreased the elevated levels of N1-acetylspermidine to control values in the distal colons of animals treated with carcinogen for 15 and 26 weeks; and (5) difluoromethylornithine markedly reduced the number of tumors induced by dimethylhydrazine in the distal but not proximal colonic mucosa at 26 weeks.     

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.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Secretin-induced accumulation of N1-acetylspermidine and putrescine in the rat pancreas

The effects of secretin on polyamine metabolism in rat pancréas were investigated. Single injections of secretin increased ornithine decarboxylase activity only very slightly. However a substantial time- and dose-dependent increase of acetyl CoA: polyamine N¹-acetyltransferase activity was observed. The concentrations of N¹-acetylspermidine, putrescine and β-alanine increased concomitantly, but spermidine and spermine remained unchanged. These results suggest that, in this model, the accumulated putrescine was formed from spermidine, via its acetylation, rather than from ornithine.     

Metabolic and antiproliferative consequences of activated polyamine catabolism in LNCaP prostate carcinoma cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.     

Genetically altered expression of spermidine/spermine N1-acetyltransferase affects fat metabolism in mice via acetyl-CoA

The acetylating enzyme, spermidine/spermine N1-acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N1-acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N1-acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N1-acetyltransferase knock-out mice. Tissues of the former were characterized by increased N1-acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl- and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N1-acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl- and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N1-acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl- and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.     

Acetylation of polyamines in chicken brain and retina

Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration.     

Effect of carbon tetrachloride on polyamine metabolism in rodent liver

Administration of hepatotoxic doses of carbon tetrachloride to mice produced a 25-fold increase in spermidine/spermine N¹-acetyltransferase activity within 6 h, but did not significantly change the activity of polyamine oxidase. The content of acetylated polyamines in the mouse liver was increased more than 100-fold from levels below the limit of detection to 0.6 μmol of N¹-acetylspermidine and 0.045 μmol of N¹-acetylspermine per gram of tissue. Putrescine levels also rose by 7-fold within 6 h and by 21-fold within 24 h. These results are in contrast to changes in hepatic polyamines brought about in the rat by carbon tetrachloride. Although the hepatotoxin produced a similar increase in spermidine/spermine N¹-acetyltransferase in this species, the rise in acetylated polyamines was much smaller and more transient. The content of N¹-acetylspermidine was increased only to 0.066 μmol/g and N¹-acetylspermine was not detected. However, in the rat putrescine increased 35-fold within 6 h and 64-fold by 16 h. These differences appear to be due to the much higher polyamine oxidase activity which was 20 times greater in the rat than in the mouse liver. This oxidase converts N¹-acetylspermine to spermidine and degrades N¹-acetylspermidine to putrescine. Spermine content was significantly reduced in both species after exposure to carbon tetrachloride, but only part of this decline could be attributed to the increased acetylation.     

Comparison of 3-isobutyl-1-methylxanthine-induced accumulation of putrescine in rat pancreas and liver

3-Isobutylmethylxanthine (IBMX), a potent phosphodiesterase inhibitor, causes accumulation of putrescine of same magnitude in rat pancreas and liver. IBMX produces increases of acetyl CoA: polyamine N'-acetyltransferase (PAT) and of ornithine decarboxylase (ODC) activities in both organs. However ODC activity is 300 times higher in liver than in pancreas. In the latter organ, there is a transient increase of N1-acetylspermidine, followed by a decrease of spermidine, alpha-Difluoromethylornithine (DFMO), a potent ODC inhibitor, impairs the accumulation of putrescine in liver but not in pancreas. These results suggest that in pancreas the accumulated putrescine is essentially formed from spermidine, via N1-acetylation and oxidation, while in liver it is formed from decarboxylation of ornithine. A possible involvement of cAMP in the stimulation of the polyamine interconversion pathway is discussed.     

Excretion of acetylated and free polyamines by polyamine depleted Chinese hamster ovary cells

1. Cultured Chinese hamster ovary cells (CHO) and their ornithine decarboxylase deficient mutant cells (C55.7) were found to excrete small amounts of N8-acetylspermidine and free polyamines, putrescine and spermidine into the culture medium. 2. The concentration of N8-acetylspermidine in the control cells was 2-3% of that of spermidine. In the medium, however, the amount of N8-acetylspermidine was about 2-fold that of spermidine and 2- to 3-fold higher than the intracellular amount. N1-acetylspermidine or acetylated spermine were never detected in the cells or in the media. 3. Confluent CHO cells treated with 2 mM difluoromethylornithine stopped the excretion when the intracellular spermidine concentration had decreased to 20% of control while there was no decrease in spermine concentration. At low cell density, neither polyamine depleted CHO cells nor the C55.7 cells excreted any polyamines into the culture media.     

Genetic manipulation of polyamine catabolism in rodents

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Chronic lithium treatment prevents the dexamethasone-induced increase of brain polyamine metabolizing enzymes.

The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithium's action and in turn provide new avenues for exploring polyamine function in the brain.     

Spermine oxidase SMO(PAOh1), Not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.     

Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1 alpha,25-dihydroxyvitamin D3-induced putrescine synthesis

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.     

Polyamine homeostasis in arginase knockout mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N¹-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues. ornithine; putrescine; spermidine; spermine; decarboxylase