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The role of polyamines in carbon tetrachloride (CCl(4))-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl(4) 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 CCl(4), 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 CCl(4) 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, alpha-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 CCl(4) induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl(4)-induced hepatic and pancreatic damage and promotes tissue regeneration.

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.

Polyamines are essential for normal cellular growth and function. Activation of polyamine catabolism in transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase, the key enzyme in polyamine catabolism, results in severe acute pancreatitis. Here, we investigated the role of polyamine catabolism in pancreatitis and studied the effect of polyamine analogues on the outcome of the disease. Polyamine depletion was associated with arginine- and cerulein-induced pancreatitis as well as with human acute necrotizing and chronic secondary pancreatitis. Substitution of depleted polyamine pools with methylspermidine partially prevented arginine-induced necrotizing pancreatitis whereas cerulein-induced edematous pancreatitis remained unaffected. Transgenic rats receiving methylated polyamine analogues after the induction of pancreatitis showed less pancreatic damage than the untreated rats. Most importantly, polyamine analogues dramatically rescued the animals from pancreatitis-associated mortality. Induction of spermidine/spermine N(1)-acetyltransferase in acinar cells isolated from transgenic rats resulted in increased trypsinogen activation. Pretreatment of acini with bismethylspermine prevented trypsinogen activation, indicating that premature proteolytic activation is one of the effects triggered by polyamine depletion. Our data suggest that activation of polyamine catabolism is a general pathway in the pathogenesis of acute pancreatitis and that experimental disease can be ameliorated with stable polyamine analogues.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

We recently generated a transgenic rat model for acute pancreatitis, which was apparently caused by a massive depletion of pancreatic polyamines spermidine and spermine due to inducible activation of their catabolism (Alhonen, L., Parkkinen, J. J., Keinänen, T., Sinervirta, R., Herzig, K. H., and Jänne, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8290-8295). When subjected to partial hepatectomy, these animals showed striking activation of polyamine catabolism at 24 h postoperatively with a profound decrease in hepatic spermidine and spermine pools and failure to initiate liver regeneration. Here we show that pancreatitis in this model could be totally prevented, as judged by histopathology and plasma alpha-amylase activity, by administration of 1-methylspermidine, a metabolically stable analogue of spermidine. Similarly, the analogue, given prior to partial hepatectomy, restored early liver regeneration in the transgenic rats, as indicated by a dramatic increase in the number of proliferating cell nuclear antigen-positive hepatocytes from about 1% to more than 40% in response to the drug. The present results suggest that the extremely high concentration of spermidine in the pancreas, in fact the highest in the mammalian body, may have a critical role in maintaining organ integrity. The failure to initiate liver regeneration in the absence of sufficient hepatic polyamine pools similarly indicates that polyamines are required for proper commencement of the regenerative process.

A large number of studies applying inhibitors of polyamine biosynthesis have indicated that these compounds are required for animal cell proliferation. Here we show, using a transgenic rat model with activated polyamine catabolism, that a certain critical concentration of the higher polyamines spermidine and spermine is required for liver regeneration. Partial hepatectomy of transgenic rats expressing spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of mouse metallothionein promoter strikingly induced the enzyme at 24 h and reduced hepatic spermidine by 80%. At that time, the weight of the liver remnant was significantly increased in syngenic rats and proliferating cell nuclear antigen (PCNA) labelling index was 20%, whereas the transgenic rats showed no liver weight gain and their PCNA-positive cells accounted for 0.5% of hepatocytes. Similarly, hepatic thymidine incorporation was markedly enhanced at this time point in syngenic, but not in transgenic, animals, whereas the rate of leucine incorporation was only marginally affected in the transgenic animals. At 3 days after operation, the spermidine pool in transgenic livers had increased to the pre-operative level, the remnant weight was significantly elevated and hepatic PCNA labelling index increased to 5%. N(1),N(11)-Diethylnorspermine, a powerful inducer of SSAT, inhibited liver weight gain and proliferative activity in both syngenic and transgenic rats. We found an extremely close correlation between hepatic spermidine, and less close between spermine, concentrations and PCNA labelling index during early liver regeneration. These results indicate that spermidine and/or spermine, but apparently not putrescine, are required for liver regeneration, yet at concentrations smaller than those normally found after partial hepatectomy.

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine/spermine N(1)-acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N(1)-acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N(1),N(2)-bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N(1),N(11)-diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N(1), N(11)-diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.

We have generated a transgenic mouse line harbouring the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter. Even in the absence of an exposure to heavy metals, ornithine decarboxylase was over-expressed in heart, testis, brain, and especially in liver, of the transgenic animals. An exposure of the transgenic mice to zinc further enhanced the enzyme activity to a level which in liver represented up to 8000-fold increase in comparison with non-transgenic animals. The striking stimulation of liver ornithine decarboxylase activity upon treatment of the transgenic mice with zinc was accompanied by a nearly 150-fold increase in the hepatic putrescine content as compared with similarly treated non-transgenic animals. Even though the liver putrescine concentration reached that of spermidine and spermine in the transgenic animals, the contents of the higher polyamines only transiently increased upon zinc administration and then returned to the basal level. These findings once again indicate that mammalian cells possess extremely powerful regulatory machinery to prevent an over-accumulation of spermidine and spermine in non-dividing cells, and that very high tissue putrescine concentrations can be tolerated, at least for periods of a few days, with seemingly no phenotypic changes.

Oxidized spermine, an iminoaldehyde (N,N'-bis (3-propionaldehyde) 1,4-diaminobutane), is a non-competitive inhibitor of fructolysis by human spermatozoa. The inhibition constant is about 0.3 mM. In experiments with [U-14C]fructose the iminoaldehyde caused a more pronounced depression of the formation of CO2 than of lactate. The iminoaldehyde was without influence on the conversion of fructose to lactate by cell-free extracts of spermatozoa, but it markedly decreased the uptake of fructose and lactate by spermatozoa. These findings strongly suggest that inhibition of the fructose metabolism of intact spermatozoa was due to interaction of the iminoaldehyde with sperm membranes and not to inhibition of any enzyme of the glycolytic pathway. Several aliphatic and aromatic aldehydes were also tested for their ability to inhibit sugar utilization of human spermatozoa: only n-hexanal exerted an inhibitory effect, the extent of which approached that of oxidized spermine.

Gossypol, a polycyclic compound isolated from cotton seeds, had a dose-dependent inhibitory effect on human sperm motility. The drug also inhibited powerfully fructolysis and glycolysis by human spermatozoa. Both lactate and CO2 formation from the 14C-labelled sugars was inhibited, and the prevention of CO2 formation from [1-14C]pyruvate and [2-14C]pyruvate by gossypol indicated a direct effect on the tricarboxylic acid cycle. Repeated washing of the sperm cells after gossypol pretreatment failed to abolish the inhibitory effect on CO2 production. The profound disturbances of the sperm energy metabolism induced by gossypol were also reflected by a striking fall of the sperm ATP content. Gossypol had little effect on glucose utilization by minces of human vaginal mucosa, indicating the specificity of gossypol.

Both 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), and methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), strikingly stimulated melanotic expression of murine Cloudman S91 melanoma cells. The stimulation of tyrosinase (EC 1.10.3.1) activity and melanin formation by DFMO was closely associated with intracellular depletion of putrescine and spermidine developed in response to the drug. However, little or no evidence was obtained indicating that enhanced melanogenesis in response to MGBG was mediated through an inhibition of polyamine biosynthesis. Indirect inhibitors of ornithine decarboxylase, such as 1,3-diaminopropane and 1,3-diaminopropan-2-ol, but not putrescine, likewise inhibited the growth of the melanoma cells and stimulated their melanin production. The stimulation of melanogenesis by polyamine antimetabolites was not mediated by cyclic adenosine 3':5'-monophosphate, in contrast to the effect elicited by alpha-melanotropin. It is also unlikely that MGBG or the diamines acted as lysosomotropic agents capable of stimulating tyrosinase activity in situ, since the enzyme activity was stimulated by the drugs irrespective of whether assayed in cultured cells or using cell-free homogenates. None of the agents stimulated tyrosinase activity in vitro. The effect of DFMO and MGBG on melanoma cell proliferation was reversible, but the restoration of normal growth and melanin formation, especially in cells exposed to DFMO, was remarkably slow. The present results represent a further experimental model, in which the inhibition of polyamine accumulation is accompanied by signs of terminal differentiation.