Ornithine decarboxylase activity and polyamine content of normal and fluoride resistant LS cells.

The activity of ornithine decarboxylase (ODC) in suspension cultures of mouse fibroblasts, LS cells, varied in a characteristic cyclic pattern after dilution of the cultures. Two strains of fluoride resistant LS cells, FR6 and LSFR6 cells, exhibited the same cyclic pattern, but with markedly higher ODC activities. These fluoride resistant cells, however, contained less putrescine, the product of the ODC reaction. Possible reasons for this finding are discussed.     

Prolonged, intravenous paraquat infusion in the rat. II. Paraquat-induced alterations in lung polyamine metabolism

The effects of paraquat (PQ) on lung putrescine, spermidine, and spermine levels, and ornithine decarboxylase (ODC) activity were assessed in rats after 7 days of iv infusion of the herbicide via osmotic minipump. Paraquat administration at a rate of 250 nmol/hr [673 +/- 40 nmol/kg/hr (n = 15)] had no effect on these parameters. In contrast, significant (p less than 0.05) elevations in lung putrescine (407% of control), spermidine (202% of control), and ODC activity (174% of control were measured in lungs of rats given 500 nmol PQ/hr [1.31 +/- 0.53 mumol/kg/hr (n = 14)]. Since evidence of lung damage was, likewise, observed only in the high-dose PQ rats, these changes in polyamine metabolism could have been a nonspecific response to PQ-induced lung injury rather than a direct biochemical effect of PQ. The results suggest that stimulation of polyamine biosynthesis may play an important role in PQ-induced lung injury. This role may involve regulation of repair mechanisms or, conversely, the polyamines may actually mediate PQ-induced fibrotic changes in the lung.     

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 microM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 microM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 microM N-acetyl putrescine, N-acetyl serotonin or N-omega-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistamic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55 microM 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N-N distance (6A) and the charge of one amino group are important chemical characteristics for regulatory effects.     

Prolactin-induced polyamine biosynthesis in spleen and thymus: specific inhibition by cyclosporine

The induction of ornithine decarboxylase (ODC) in the rat spleen and thymus in response to prolactin shows a dose-dependent sensitivity to cyclosporine (CsA), a known immunosuppressive drug. Marked inhibition of prolactin-stimulated ODC activity occurred at 0.12 mg CsA/kg body weight, and nearly total inhibition was detected at 1.2 mg CsA/kg, a dose comparable to that used to suppress organ rejection processes. CsA blocked ODC induction in response to prolactin injection in both intact and hypophysectomized rats, suggestive of a direct effect of prolactin on spleen and thymus. Furthermore, we were able to demonstrate specific 125I-prolactin binding sites on the rat spleen and thymus. Although growth hormone was capable of elevating ODC activity in spleen, this elevation was not inhibited by CsA. ODC activity in response to cyclic AMP-mediated hormones and to steroid analogs such as dexamethasone was not affected by CsA. Agents known to alter calcium channeling mostly had random enhancing capacity when administered with CsA. The most potent elevation occurred in spleens in response to aminophylline plus CsA. It should be noted, however, that aminophylline alone slightly inhibited the basal level of ODC in the spleen. Insulin, which elevated ODC in the thymus and spleen in a dose-dependent manner, also was not affected by concurrent CsA administration. Prolactin administration altered the RNA and DNA content of spleen and thymus indicating its ability to alter metabolic function. We conclude that prolactin may regulate immune function in spleen and thymus. Studies are in progress to identify the regulation of specific gene products by prolactin.     

Effects of alpha-difluoromethylornithine on polyamine biosynthesis and tyrosine hydroxylase induction in the adrenal gland of the rat subjected to stress or apomorphine

Ornithine decarboxylase (ODC) and tyrosine hydroxylase (TH), the first enzymes in the polyamine and catecholamine biosynthetic pathways, respectively, are induced in the adrenal gland of the rat through the application of stressors or dopamine agonists. In the present work, following exposure of rats to cold, application of bodily restraint, or administration of apomorphine (APM), adrenal putrescine increased in proportion to the induction of ODC. Spermidine content increased by 60% after APM and about 30% after immobilization. Spermine was unaffected. To test whether the increases of ODC (and polyamines) are necessary to the slower and later induction of TH, induction of ODC in vivo was undertaken. alpha-Difluoromethylornithine (alpha-DFMO), an irreversible inhibitor of ODC, given orally or subcutaneously, almost completely abolished the induction of ODC by APM or immobilization, and inhibited the increase of putrescine in both cases, but did not affect spermidine after APM. Repeated administration of alpha-DFMO over several days did not affect the induction of adrenal TH. The results question whether increases of adrenal ODC activity and of putrescine are essential for the induction of TH in that gland.     

Ornithine decarboxylase and spermidine/spermine N1-acetyltransferase are induced in K562 cells by S-adenosylmethionine decarboxylase inhibitor methylglyoxal bis(guanylhydrazone) but not by analogous methylglyoxal bis(butylamidinohydrazone)

The activities of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT) were increased by the addition of S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor methylglyoxal bis(guanylhydrazone) (MGBG) in cultured human erythroid leukemia K 562 cells. ODC activity began to increase 4 hr after the addition of the drug and attained a maximum at 12 hr. The increase of SAT activity lagged behind that of ODC activity. The increases of both ODC and SAT activities produced by MGBG were blocked by treatment with cycloheximide, suggesting that the increase of enzyme activity resulted from the synthesis of new enzyme proteins. The putrescine content in cells treated with MGBG increased markedly, whereas the levels of spermidine and spermine were depressed lower. On the other hand, methylglyoxal bis(butylamidinohydrazone) (MGBB), a derivative of MGBG inhibiting AdoMetDC effectively, did not induce ODC or SAT activities.     

Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.     

Hexachlorobenzene-induced early changes in ornithine decarboxylase and protein tyrosine kinase activities, polyamines and c-Myc, c-Fos and c-Jun proto-oncogenes in rat liver.

Hexachlorobenzene (HCB) is a lipophilic chemical compound that is widely distributed in the environment. HCB is known to cause liver tumors in experimental animals. In the present study the in vivo effect of HCB treatment on ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) activities, free polyamine content, and c-Myc, c-Fos, and c-Jun protein levels in rat liver were investigated. HCB (1000 mg/kg body weight) increased hepatic immunodetectable c-Myc, c-Fos, and c-Jun levels after 6 h, and ODC activity and spermine and putrescine content after 18 and 24 h, while maximum stimulation of PTK activity occurred at 12 h. PTK and ODC activities varied in a dose-dependent manner. The time-course of c-Myc, c-Fos, and c-Jun protein levels was different for each proto-oncogene. They were all elevated at the second day of treatment, while only c-Fos and c-Jun remained elevated after 10 days of HCB exposure. These data jointly suggest that the increase in ODC activity may be the consequence of proto-oncogene induction. The alterations in PTK activity suggest that the growth factor signal transduction pathway may be involved in the regulation of the proto-oncogene levels or/and ODC activity. The decrease in PTK activity after the first day, even in the presence of alpha-D-Difluoromethylornithine (DFMO), an inhibitor of ODC activity, suggests that it is not regulated by polyamines. These results may be relevant to the early molecular events involved in HCB tumor promoter activity in rat liver.     

Inhibition by polyamines of methylthiopropylamine-induced ornithine decarboxylase in human lymphoid leukemia Molt 4B cells

Methylthiopropylamine (MTPA), an inhibitor of spermidine synthase, markedly induced ornithine decarboxylase (ODC) activity (about 30-fold of the basal level) in human lymphoid leukemia Molt 4B cells. This induction was blocked by the addition of spermidine, spermine or putrescine simultaneously with MTPA. Inhibition by spermidine or spermine of the MTPA-induced ODC activity was larger than that by putrescine. The increase of ODC activity by MTPA led to the large increase of cellular putrescine content. This increase of putrescine content was abolished drastically by the simultaneous addition of spermidine or spermine. The increase of ODC activity was almost completely blocked by the addition of cycloheximide or actinomycin D. This finding suggested that the increase of ODC activity was not due to activation of ODC preformed in Molt 4B cells. The ODC induction by MTPA was dose-dependently blocked by adding the calcium channel blockers (verapamil and nifedipine) or protein kinase C inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine and palmitoyl carnithine). These results suggested that calcium and protein kinase C (PKC) were involved in MTPA-associated induction of ODC.     

Ornithine Decarboxylase Activity and Polyamine Content of Normal and Fluoride Resistant LS Cells

Abstract The activity of ornithine decarboxylase (ODC) in suspension cultures of mouse fibroblasts. LS cells, varied in a characteristic cyclic pattern after dilution of the cultures. Two strains of fluoride resistant LS cells, FR6 and LSFR6 cells, exhibited the same cyclic pattern, but with markedly higher ODC activities. These fluoride resistant cells, however, contained less putrescine, the product of the ODC reaction. Possible reasons for this finding are discussed.     

Changes in polyamine metabolism in rat liver after oral administration of alpha-naphthylisothiocyanate

Changes in the levels of urea cycle enzymes and polyamine metabolism in the liver of rats treated with alpha-naphthylisothiocyanate (ANIT), an inducer of experimental cholestasis, were studied. Activities of arginase increased approximately two-fold compared to the control values during the period of 24-72 hours after oral administration of ANIT (100 mg/kg), while activities of ornithine carbamyltransferase and ornithine aminotransferase decreased. The activity of ornithine decarboxylase was elevated by approximately 20- and 10-fold at 12 and 60 hours, respectively, after ANIT administration. Putrescine concentration doubled 24-48 hours after the ANIT administration, but spermidine level rose more slowly and reached the level of 1.5-fold of the control level in 36-72 hours. Spermine concentration decreased initially but increased in 96 hours. These results suggest that the increased activity of urea cycle accounts for the increase in the ornithine content and that the putrescine and spermidine acts as the initiator of recovery of the liver damaged by ANIT treatment.     

Suppression of lymphocyte proliferation by hexamethylene diamine

The antiproliferative potential of hexamethylene diamine (HMDA) for mitogen-stimulated splenic lymphocytes was evaluated in vitro at final concentrations of 0.1-16 mM. Addition at the start of culture or after 24 or 48 h of culture decreased the proliferative response to T and B cell mitogens. However, the concentration of HMDA required to cause suppression increased with incubation time. Removal of diamine after 24 h allowed cells to proliferate normally upon reculture with mitogen. Mitogenic responses of cultures containing the potent ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were also inhibited in a time and dose dependent fashion. ODC activity, which was much greater in cultures stimulated with Con A than LPS, was markedly decreased by inclusion of diamine or DFMO in the culture medium. Addition of putrescine to cultures did not reverse the suppressive effects of diamine on proliferation but did restore DFMO-containing cultures to control levels of activity. These results indicate that HMDA does suppress lymphocyte proliferation in vitro by alteration of ODC and polyamine activity. However, comparison of results obtained with DFMO and HMDA suggests that HMDA may act via multiple pathways, only one of which involves inhibition of ODC activity.     

Immunological studies on sporamycin-treated animals

Sporamycin showed a remarkable tumor regressive activity against sarcoma-180 with a single 5 mg/kg dose of intravenous administration. This antitumor effect on tumor and host animals was examined immunologically. As the results: (1) When sarcoma-180 tumor cells were used as an antigen macrophage migration inhibition reaction by spleen cells derived from the tumor-bearing mice treated with sporamycin was positive at day 7 approximately 14 after the medication and was negative thereafter. (2) The delayed hypersensitivity tested by the foot-pad reaction was positive in tumor-bearing mice treated with sporamycin, and no decrease of foot pad reaction was observed, whereas this reaction decreased remarkably in non-treated tumor-bearing mice. (3) Sarcoma-180 tumor cells were mixed with spleen cells derived from sporamycin-treated mice, and were inoculated into normal dd mice. The growth of tumor cells was inhibited markedly, but no inhibition of tumor growth was observed in case of spleen cells derived from non-treated tumor bearing mice. (4) Combined treatment of sporamycin with PS-K, an immunopotentiator, showed a remarkable synergistic effect.     

Inhibition of murine embryonic development by α-difluoromethylornithine,an irreversible inhibitor of ornithine decarboxylase

The activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMDC) and the concentrations of putrescine, spermidine and spermine were measured in mouse uterus, placenta and foetus during gestation. The prominent post-implantation biochemical changes in the intact uterus were associated mainly with the deciduomata and significant ODC activity was located in theembryo. Administration of the irreversible inhibitor of ODC, α-difluoromethylornithine, DFMO, 2% in the drinking water during days 5–8 of gestation, abolished the increases in uterine ODC activity, putrescine and spermidine concentrations and enhanced the activity of SAMDC. Treated animals showed no signs of pregnancy when autopsied on day 18. The alterations in deciduomal weight and the changes in uterine DNA, RNA and protein content indicated that decidualization following DFMO took place normally but that embryonic growth was arrested. Treatment on single days with DFMO, 200 mg/kg every six h, revealed optimal contragestational effects on day 8 which corresponded exactly to the time of the peak in deciduomal ODC activity. Treatment with DFMO at times other than during the vulnerable period of days 5–8 has less prominent effects on gestation. An increase in ODC activity appears to be an essential factor during a short, but critical, period after implantation for continued murine embryonal growth.     

Role of polyamines in myocardial ischemia/reperfusion injury and their interactions with nitric oxide

Polyamines (putrescine, spermidine, and spermine) are present in all higher eukaryotic cells and are essential for cell growth, differentiation and apoptosis. Sharing common precursor with polyamines, nitric oxide (NO) is associated with myocardial ischemia/reperfusion injury by the generation of peroxynitrite. Although polyamines have been implicated in tissue ischemia injury, their metabolism and interactions with NO in myocardial ischemia/reperfusion injury have not been fully understood. In our experiment, when Langendorff perfused rat hearts were subjected to 40 min ischemia without reperfusion, both ornithine decarboxylase (ODC) and Spermidine/spermine N(1)-acetyltransferase (SSAT) activities were up-regulated and putrescine accumulated. While after reperfusion, ODC activity decreased and SSAT activity increased, resulting in putrescine accumulation and decreased spermidine and spermine. Meanwhile NO content was increased. In addition, sodium nitroprusside (SNP, a NO donor) decreased ODC activity in cardiac tissue homogenate but increased SSAT activity in a dose-dependent manner. Pre-treatment of isolated heart with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NO synthase) increased ODC activity. Exogenous spermine (1 mM) administration prior to ischemia prevented spermine decrease, reduced cardiac myocyte necrosis and apoptosis, and promoted the recovery of cardiac function after ischemia/reperfusion. These results suggest that acute heart ischemia activates myocardial polyamine stress response characterized by increased ODC and SSAT activities and accumulation of putrescine. Ischemia/reperfusion disturbs polyamine metabolism, and the loss of spermine might be associated with NO increase and thereby influences myocardial cell viability. Exogenous spermine may protect the hearts from myocardial ischemia/reperfusion injury.     

Stabilization of ornithine decarboxylase in mouse liver and lung by methylglyoxal bis(cyclohexylamidinohydrazone)

The intraperitoneal injection of methylglyoxal bis(cyclohexylamidinohydrazone) (MGBC), an inhibitor of S-adenosylmethionine decarboxylase and spermidine synthase, markedly increased (7-fold of the basal level at 4 hr) ornithine decarboxylase (ODC) activity in normal mouse liver. ODC activity was also increased 2.5-fold over the basal level in mouse lung at 6 hr after the injection. The effect of MGBC on ODC activity occurred in a dose-dependent manner. Measurement of the apparent half-life of ODC induced in the liver and lung by MGBC treatment revealed a clear decrease in the decay rate of the enzyme in both the tissues. Activities of S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine/spermine N1-acetyltransferase (SAT) were not increased by the intraperitoneal injection of MGBC. There was a large rise in putrescine and a fall in spermidine and spermine in the liver and lung except for brain within an 8 hr period in response to MGBC, suggesting that these changes resulted from the stabilization of ODC and inhibitions of AdoMetDC and spermidine synthase.     

Induction of Epidermal and Hepatic Ornithine Decarboxylase by a Prudhoe Bay Crude Oil

Induction of Epidermal and Hepatic Ornithine Decarboxylase bya Prudhoe Bay Crude Oil. RAHIMTULA, A. D., LEE, Y.-Z. and SILVA,J. (1987). Fundam. Appl. Toxicol. 8, 408–414. Applicationof a Prudhoe Bay crude oil (PBCO) to the backs of mice causeda rapid induction of epidermal ornithine decarboxylase (ODC).A maximum induction of over 60-fold was seen at 6 hr after application of 50 µl of PBCO. Concurrently, epidermal putrescinelevels were elevated 4.7-fold over controls. Intraperitonealadministration of PBCO led to a 15-20-fold increase in hepaticODC activity but to a 45% decrease in the renal enzyme activity.Maximum induction of ODC was seen at 12 hr following the administrationof 4 ml/kg body wt of PBCO. Hepatic putrescine levels were elevated34-fold over controls. Pretreatment of mice with the proteinsynthesis inhibitor cycloheximide abolished both epidermal andhepatic ODC induction.     

Effect of adrenergic stimulation on ornithine decarboxylase activity in the rat spleen

The effect of a single administration of catecholamines on ornithine decarboxylase activity and polyamine biosynthesis in the rat spleen was investigated. Isoproterenol elicited a dose-dependent increase in spleen ODC activity which reached a maximum 4 hr after the administration of the drug. Putrescine content was also found to increase within a few hours, whereas S-adenosylmethionine decarboxylase activity and spermidine and spermine levels did not change significantly. Adrenaline and noradrenaline proved to be even more effective in increasing splenic ODC activity than isoproterenol. alpha- and beta-adrenergic antagonists prevented the ODC increase by catecholamines to a different extent.     

Increase in histidine decarboxylase activity in tissues of mice bearing Colon-26 tumor cells

The changes in histidine decarboxylase (HDC) activity, histamine and tele-methylhistamine contents were examined in tissues of mice after the inoculation of Colon-26 tumor cells subcutaneously into the lower back. The HDC activity in the spleen of mice increased significantly 14 days after the inoculation of Colon-26 and the increase in HDC activity continued for up to 28 days. However, the histamine content in the spleen of tumor-bearing mice was not changed significantly during the observation period. In the following experiments, two subclones of the Colon-26 cell line, cachexia-inducing clone-20 and non cachexia-inducing clone-5, were used and the induction of HDC activity in mice was examined in four tissues, spleen, lung, liver and kidney. Both clone-20 and clone-5 induced the increase in HDC activity to the same extent in the spleen and lung, but not in the liver and kidney. As observed using the Colon-26 original cell line, the histamine contents in the four tissues of tumor-bearing mice were not different from those in the control mice. In contrast, the levels of tele-methylhistamine, one of the major catabolites of histamine, in the tumor-bearing mice increased significantly compared with the control mice in all four tissues examined. There was a correlation between the increase in tele-methylhistamine level and the increase in HDC activity in the tissues. A histological study indicated that the tissue mast cells were not increased in spleen and lung of tumor-bearing mice. These findings indicated that the increase in HDC activity in the spleen and lung occurred in parallel with the growth of inoculated tumor cells in mice and suggested that the cells other than mast cells may be involved in the increase in HDC activity. The tumor-bearing state produced histamine with a high turnover rate in the mouse tissues, especially in the spleen and lung. Received: 30 November 1998 / Accepted: 14 April 1999 / Published online: 2 July 1999