Effects of MDL 72527, a Specific Inhibitor of Polyamine Oxidase, on Brain Edema, Ischemic Injury Volume, and Tissue Polyamine Levels in Rats After Temporary Middle Cerebral After Occlusion

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex ( P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex ( P < 0.05) and 17% in the subcortex ( P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia-reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.     

Inhibition of polyamine oxidase in rats improves the sensitivity of urinary polyamines as markers for cell death.

In this study we investigated polyamine metabolism during inhibition of two polyamine-catabolizing enzymes. This was performed by treating rats with aminoguanidine [an inhibitor of Cu-dependent amine oxidase (CuAO)], NN'-bis(buta-2,3-dienyl)butane-1,4-diamine [MDL 72527, an inhibitor of FAD-dependent polyamine oxidase (PAO)], tetrachloromethane (hepatotoxic agent) and combinations of these compounds. Emphasis was laid on the origin and possible clinical usefulness of two polyamine metabolites: acetylisoputreanine-gamma-lactam and N1N12-diacetylspermine. Acetylisoputreanine-gamma-lactam is a normal constituent of human and rat urine. Treatment of rats with aminoguanidine led to undetectable urinary levels of acetylisoputreanine-gamma-lactam, whereas MDL 72527 treatment resulted in a 12-fold increase. Under normal conditions this compound represents a minor CuAO catabolite of N1-acetylspermidine, but may become of more importance under CuAO-induced conditions. N1N12-diacetylspermine was undetectable in urine samples from non-pregnant adults and rats, but became detectable after treating rats with MDL 72527. Additional tetrachloromethane poisoning resulted in a 35-fold increase of N1N12-diacetylspermine in urine and its appearance in liver. Hence urinary excretion of N1N12-diacetylspermine during PAO inhibition may serve as a sensitive marker for cell death. This was confirmed by myeloid-leukaemia-bearing rats treated with MDL 72527, which also excreted N1N12-diacetylspermine in urine in relatively high amounts from at least day 14 until spontaneous death.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Effect of the polyamine oxidase inactivator MDL 72527 on N(1)-(n-octanesulfonyl)spermine toxicity

N(1)-(n-octanesulfonyl)spermine (N(1)OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N(1)OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N(1)OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N(1)OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N(1), N(12)-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N(1)OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N(1)OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.     

Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role

N1-Methyl-N2-(2,3-butadienyl)-1,4-butanediamine (MDL 72521) and N1,N2-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) are specific, potent, enzyme-activated, irreversible inhibitors of polyamine oxidase in vitro. These compounds are also capable of completely inhibiting polyamine oxidase in mouse tissues at intraperitoneal doses greater than 20 mg/kg. Enzyme activity reappears in the various organs within 2-3 days to 50% of the control values. Irreversible inhibition of polyamine oxidase in mice led to decreased putrescine (30-40%) and spermidine (10-20%) levels in liver and some other organs. At the same time N1-acetylspermidine and, to a lesser extent, N1-acetylspermine were accumulating at rates which are assumed to be related to the rates of polyamine degradation. Even after treatment with polyamine oxidase inhibitors over a period of 6 weeks at doses which produced complete inhibition of polyamine oxidase in all organs, including the brain, neither toxic effects nor changes in body weight or behaviour were observed.     

Polyamine reutilization and turnover in brain

N¹, N²-bis-(2, 3-butadienyl)-1, 4-butanediamine (MDL 72527) is an irreversible, specific inhibitor of polyamine oxidase, which allows one to completely inactivate this enzyme in all organs of an experimental animal. As a result one observes a linear increase of N¹-acetylsperimidine and N¹-acetylspermine concentrations in brain. The rate of accumulation seems directly proportional to the rate of spermidine, and spermine degradation respectively, and since no compensatory changes of the polyamine synthetic enzymes, were induced by inhibition of polyamine oxidase, the rate of acetyl-polyamine accumulation is assumed to be a measure for polyamine turnover. The decrease of brain putrescine levels by 70 percent in the brains of MDL 72527-treated animals suggests the quantitative significance of putrescine reutilisation. Pretreatment of the animals with D, L--difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase reduced both, polyamine turnover rate and the extent of putrescine reutilization. Inhibition of GAPA-T produced a significant increase of polyamine turnover in brain, in agreement with the known induction of ornithine decarboxylase activity after treatment with inhibitors of GABA-T.     

多胺及其代谢产物对脑缺血的影响与治疗

多胺(Polyamines)是直链多价阳离子碱性胺,包括腐胺(putrescine,PUT),精胺(spermine,SPM),精脒(spermidine,SPD)等。广泛存在于各种组织细胞内,是一种代谢调控物质,在细胞的增殖分化中起着重要作用。脑梗死是成人致残、致死的最常见疾病之一。研究表明,脑缺血后,多胺及其代谢产物增加,能引起梗死面积的扩大及缺血半暗带神经细胞的坏死。其潜在机制尚不明确,可能与缺血后多胺代谢产生腐胺,3-氨基丙醛(3-amidopropanal 3-AP),过氧化氢及丙烯醛等的活性物质有关,它们参与开放钙离子通道,破坏血脑屏障,形成血管源性脑水肿及缺血再灌注性神经性损伤等病理过程。而抑制多胺代谢可有效地缓解缺血后多胺及其代谢产物增加引起的神经损伤。本文就多胺及代谢产物对脑缺血的神经毒性作用及药物抑制多胺代谢治疗脑梗死做一综述。     

On the degradation and elimination of spermine by the vertebrate organism

1. Treatment of mice and rats with the polyamine oxidase inhibitor N1,N4-bis-(2,3-butadienyl)-1,4-butanediamine (MDL 72527) causes a gradual accumulation of spermine in the circulation and a decrease of spermidine concentration. 2. Spermine is mainly localized in the red blood cells. 3. Co-administration of 2-(difluoromethyl)ornithine and MDL 72527 enhances considerably the rate and extent of spermine accumulation in the circulation. 4. It is assumed that the increased rate of spermine accumulation by the two drugs is due to the enhancement of cell death, i.e. spermine accumulation is the result of its release into the circulation from dying cells, not due to physiological release. 5. After discontinuation of polyamine oxidase inhibition spermine appears to be gradually transformed into spermidine by red blood cell polyamine oxidase, obviously without transformation into N1-acetylspermine.     

Pneumocystis carinii polyamine catabolism.

DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.     

Inhibition of polyamine and spermine oxidases by polyamine analogues

Polyamine oxidase (PAO) and spermine oxidase (SMO) are involved in the catabolism of polyamines--basic regulators of cell growth and proliferation. The discovery of selective inhibitors of PAO and SMO represents an important tool in studying the involvement of these enzymes in polyamine homeostasis and a starting point for the development of novel antineoplastic drugs. Here, a comparative study on murine PAO (mPAO) and SMO (mSMO) inhibition by the polyamine analogues 1,8-diaminooctane, 1,12-diaminododecane, N-prenylagmatine (G3), guazatine and N,N1-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527) is reported. Interestingly, 1,12-Diaminododecane and G3 behave as specific inhibitors of mPAO, values of K(i) for mPAO inhibition being lower than those for mSMO inactivation by several orders of magnitude. The analysis of molecular models of mPAO and mSMO indicates a significant reduction of the hydrophobic pocket located in maize PAO (MPAO) at the wider catalytic tunnel opening. This observation provides a rationale to explain the lower affinity displayed by G3, guazatine and MDL72527 for mPAO and mSMO as compared to MPAO. The different behaviour displayed by 1,12-diaminododecane towards mPAO and mSMO reveals the occurrence of basic differences in the ligand binding mode of the two enzymes, the first enzyme interacting mainly with substrate secondary amino groups and the second one with substrate primary amino groups. Thus, the data reported here provide the basis for the development of novel and selective inhibitors able to discriminate between mammalian SMO and PAO activities.     

α-Methylspermidine protects against carbon tetrachloride-induced hepatic and pancreatic damage

The role of polyamines in carbon tetrachloride (CCl₄)-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl₄ resulted in the induction of hepatic spermidine/spermine N ¹-acetyltransferase (SSAT), accumulation of putrescine, reduction in spermine level and appearance of moderate hepatic injury within 24 h. Upon treatment with CCl₄, transgenic rats overexpressing SSAT displayed induction of both hepatic and pancreatic SSAT, with subsequent accumulation of putrescine and decrease of both spermidine and spermine pools. Administration of CCl₄ in SSAT transgenic rats induced not only massive hepatic injury, but also severe acute necrotizing pancreatitis. Pretreatment of the animals with catabolically stable functional polyamine mimetic, α-methylspermidine (MeSpd) prevented pancreatic and hepatic injury in SSAT rats and markedly reduced liver damage in syngenic animals. As assessed by immunostaining of proliferating cell nuclear antigen, MeSpd increased the amount of regenerating hepatocytes in both genotypes. These results show that CCl₄ induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl₄-induced hepatic and pancreatic damage and promotes tissue regeneration.     

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.     

The influence of catabolic reactions on polyamine excretion.

Complete inhibition of polyamine catabolism is possible by combined administration of two compounds. Aminoguanidine (25 mg/kg body wt., intraperitoneally) inhibits all reactions that are catalysed by copper-containing amine oxidases (CuAO). The products of the CuAO-catalysed reactions cannot be reconverted into polyamines (terminal catabolism) and therefore usually escape observation. N1-Methyl-N2-(buta-2,3-dienyl)butane-1,4-diamine (MDL 72521) is a new inhibitor of polyamine oxidase. It inhibits completely the degradation of N1-acetylspermidine and N1-acetylspermine. The enhanced excretion of N1-acetylspermidine in urine after administration of 20 mg of MDL 72521/day per kg body wt. is a measure of the rate of spermidine degradation in vivo to putrescine, and thus of the quantitative significance of the interconversion pathway. From the enhancement of total polyamine excretion by aminoguanidine-treated rats, one can calculate that only about 40% of the polyamines that are destined for elimination are usually observed in the urine, the other 60% being catabolized along the CuAO-catalysed pathways. The normally observed urinary polyamine pattern gives, therefore, an unsatisfactory picture of the actual polyamine elimination. Although aminoguanidine alone is sufficient to block terminal polyamine catabolism, rats that were treated with a combination of aminoguanidine and MDL 72521 excrete more polyamines than those that received aminoguanidine alone. The reason is that a certain proportion of putrescine, which is formed by degradation of spermidine, is normally reutilized for polyamine biosynthesis. In MDL 72521-treated animals this proportion appears in the urine in the form of N1-acetylspermidine. Thus it is possible to determine polyamine interconversion and re-utilization in vivo and to establish a polyamine balance in intact rats by using specific inhibitors of the CuAO and of polyamine oxidase.     

Role of polyamine metabolism in kainic acid excitotoxicity in organotypic hippocampal slice cultures

Polyamines are ubiquitous cations that are essential for cell growth, regeneration and differentiation. Increases in polyamine metabolism have been implicated in several neuropathological conditions, including excitotoxicity. However, the precise role of polyamines in neuronal degeneration is still unclear. To investigate mechanisms by which polyamines could contribute to excitotoxic neuronal death, the present study examined the role of the polyamine interconversion pathway in kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. Treatment of cultures with N1,N(2)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527), an irreversible inhibitor of polyamine oxidase, resulted in a partial but significant neuronal protection, especially in CA1 region. In addition, this pre-treatment also attenuated KA-induced increase in levels of lipid peroxidation, cytosolic cytochrome C release and glial cell activation. Furthermore, pre-treatment with a combination of cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) and MDL 72527 resulted in an additive and almost total neuronal protection against KA toxicity, while the combination of MDL 72527 and EUK-134 (a synthetic catalase/superoxide dismutase mimetic) did not provide additive protection. These data strongly suggest that the polyamine interconversion pathway partially contributes to KA-induced neurodegeneration via the production of reactive oxygen species.     

A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase.

BACKGROUND: Polyamines are essential for cell growth and differentiation; compounds interfering with their metabolism are potential anticancer agents. Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary amino groups of spermine and spermidine. RESULTS: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Angstroms. PAO from Zea mays contains two domains, which define a remarkable 30 Angstrom long U-shaped catalytic tunnel at their interface. The structure of PAO in complex with the inhibitor MDL72527 reveals the residues forming the catalytic machinery and unusual enzyme-inhibitor CH.O H bonds. A ring of glutamate and aspartate residues surrounding one of the two tunnel openings contributes to the steering of the substrate towards the inside of the tunnel. CONCLUSIONS: PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.     

Purvalanol A is a strong apoptotic inducer via activating polyamine catabolic pathway in MCF-7 estrogen receptor positive breast cancer cells

Purvalanol A is a specific CDK inhibitor which triggers apoptosis by causing cell cycle arrest in cancer cells. Although it has strong apoptotic potential, the mechanistic action of Purvalanol A on significant cell signaling targets has not been clarified yet. Polyamines are crucial metabolic regulators affected by CDK inhibition because of their role in cell cycle progress as well. In addition, malignant cells possess impaired polyamine homeostasis with high level of intracellular polyamines. Especially induction of polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO) induced toxic by-products in correlation with the induction of apoptosis in cancer cells. In this study, we showed that Purvalanol A induced apoptosis in caspase- dependent manner in MCF-7 ER(+) cells, while MDA-MB-231 (ER?) cells were less sensitive against drug. In addition Bcl-2 is a critical target for Purvalanol A, since Bcl-2 overexpressed cells are more resistant to Purvalanol A-mediated apoptosis. Furthermore, exposure of MCF-7 cells to Purvalanol A triggered SSAT and PAO upregulation and the presence of PAO/SMO inhibitor, MDL 72,527 prevented Purvalanol A-induced apoptosis.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.