Inhibitory effects of niacin and its analogues on induction of ornithine decarboxylase activity by diethylnitrosamine in rat liver

Pharmacological doses of niacin and its analogues were given intraperitoneally to rats with and without coadministration of a hepatocarcinogenic dose of diethylnitrosamine (DEN), and their effects on the induction of ornithine decarboxylase (ODC, EC 4.1.1.17) activity in the rat liver were studied. The induction of ODC activity by DEN was inhibited by 74.3, 85.5, 94.6, 97.6, 72.6 and 55.2% by nicotinamide, nicotinic acid, 3-hydroxymethylpyridine, beta-picoline, pyridine-3-aldehyde and ethylnicotinate respectively. When given alone, these analogues did not induce ODC activity. All these compounds are known to have a niacin effect. DEN-induced ODC activity was also inhibited by 84.0, 93.3, 52.8 and 75.9% by 6-aminonicotinamide, picolinic acid, pyridine-3-sulfonic acid and thionicotinamide, respectively, but, peculiarly, they induced ODC activity by their administration alone. These niacin analogues are known to have anti-niacin effects. Tryptophan, N'-methylnicotinamide and isonicotinic acid hydrazide did not affect the DEN-induced ODC activity but could induce ODC by themselves. Tryptophan belongs to the former group and isonicotinic acid hydrazide to the latter group. The reason for these discrepancies is discussed.     

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.     

In the search for new anticancer drugs. 13. Phosphonic and phosphinic analogues of ornithine

Phosphonic (4a,b) and phosphinic (5a-d) analogues of ornithine were synthesized and evaluated for their inhibitory activity against ornithine decarboxylase and against the lymphocytic leukemia P388. The title compounds possess a low degree of inhibition against rat liver ornithine decarboxylase as compared to alpha-(difluoromethyl)ornithine. Thus, compounds 4a and 5a inhibit by 40% the ornithine decarboxylase activity at a 5 mM concentration. The other derivatives are less potent. Compounds 4a, 4b, 5b, and 5d are inactive against P388 tumor in CD2F1 mice at doses of 50 and 150 mg/kg.     

Inhibition of ornithine decarboxylase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activity of rat liver ornithine decarboxylase (ODC) were investigated. Sixteen hours after partial hepatectomy, rats that had been pretreated with TCDD for 1 week exhibited a 3- to 4-fold increase in ODC activity, while vehicle controls exhibited to 8- to 10-fold increase. This inhibition of ODC induction by TCDD was time dependent since TCDD administration at the time of partial hepatectomy did not produce inhibitory effects on the subsequent ODC induction. ODC induction after either aminophylline or dexamethasone administration, agents which act via cAMP-mediated and direct nuclear events, respectively, also was inhibited by pretreatment with TCDD. It was concluded that TCDD pretreatment decreased the ability of the liver to respond to hormonal stimulation as reflected in the attenuation of ODC induction. RNA polymerase I activity, which positively correlates with ODC activity in growth and development, decreased concomitantly with decreased induction of ODC. In unstimulated liver RNA polymerase I activity, as well as protein, DNA, and RNA levels, remained unchanged 1 week after TCDD. However, TCDD administration resulted in decreased liver concentrations of putrescine and spermidine, but not spermine. This suggests that TCDD pretreatment results in a time-dependent decrease in hormone responsivity.     

Potential inhibitors of polyamine biosynthesis. 2. alpha-Alkyl- and benzyl-(+/-)-ornithine.

Alpha-Methyl-(+/-)-ornithine hydrochloride was not a substrate for ornithine decarboxylase from rat prostate glands. It produced equal inhibition of ornithine decarboxylase obtained from rat prostate glands, spleens of mice inoculated with L1210 leukemic cells, and regenerating rat liver indicating its lack of selectivity for any of these tissues. In these three tissues the inhibition was competitive with L-ornithine. A number of alpha-alkyl- and alpha-aralkyl-substituted analogs of (+/-)-ornithine were synthesized and evaluated in vitro as inhibitors of the enzyme L-ornithine decarboxylase obtained from prostate glands of rats. These compounds were obtained by the reaction of alkyl iodide or benzyl bromide with the anion obtained by treatment of 3-(benzalimino)piperidin-2-one with sodium hydride. The following alpha-substituted analogs of (+/-)-ornithine were obtained: ethyl, n-propyl, n-butyl, n-hexyl, n-octyl, and benzyl. The synthesized compounds were found to be much less active than alpha-methyl-(+/-)-ornithine as competitive inhibitors of ornithine decarboxylase in vitro. The most active compound in the series was alpha-n-octyl-(+/-)-ornithine which was 60-fold less active than alpha-methyl-(+/-)-ornithine and the least active analog was alpha-n-butyl-(+/-)-ornithine which was 270-fold less active than the alpha-methyl-(+/-)-ornithine.     

Histamine H2-receptor mediated activation of neonatal rat brain ornithine decarboxylase in vivo

The effect of histamine (HA) administered via intracerebroventricular injection on ornithine decarboxylase (ODC) activity was studied in neonatal rat brain. The HA effect was dose and time dependent. Maximal increase in ODC activity was achieved 2 hr after administration of 10 micrograms HA (38% over control levels). Impromidine (HA H2-agonist) mimicked the effect of HA on ODC and ranitidine (HA H2-antagonist) inhibited the response to HA. Neither 2-thiazolylethylamine (HA H1-agonist) nor mepyramine (HA H1-antagonist) modified control ODC activity. The HA-releasers, compound 48/80 and polymixin B sulfate, elicited an increase in brain ODC activity of 35% and 32%, respectively, over the control value.     

Inhibition of ornithine decarboxylase by the isomers of 1,4-dimethylputrescine

1,4-Dimethylputrescine (2,5-hexanediamine) was separated into its racemic and meso isomers by fractional crystallization of its dibenzoyl derivative. The racemic form was resolved into its (+)- and (-)-isomers with (+)- and (-)-dibenzoyltartaric acids. None of the three isomers (meso, +, and -) inhibited ornithine decarboxylase (ODC) activity in vitro, while all the three were strongly inhibitory of ODC when assayed in vivo in rats or in H-35 hepatoma cells. In rat liver the three isomers also decreased the putrescine pool while only the (+)-isomer decreased spermidine content. In the H-35 cells the (-)- and (+)-isomers decreased the spermidine and spermine content. When ODC was induced in the latter by insulin it was found that the (-)-isomer strongly inhibited protein and ODC synthesis, while the (+)-isomer and the meso isomer were less inhibitory. The meso isomer was a good inducer of ODC antizyme in rat liver, while the (+)- and (-)-isomers were poor inducers of the former.     

Lack of inhibition of ornithine decarboxylase activity by ibuprofen.

The influence of the anti-inflammatory drug ibuprofen on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17), the key enzyme of polyamine synthesis, was studied using a 20,000 g supernatant of rat testis and regenerating liver homogenates as sources of the enzyme. Ibuprofen, in all concentrations studied (10(-6) to 2 x 10(-3) M), did not influence either testicular or hepatic ODC activity in vitro. The role of ODC in inflammatory processes and the lack of ODC inhibition by ibuprofen are discussed in view of the controversial findings of other authors.     

Inhibitors of polyamine biosynthesis VI: 2,5-Diamino-2-(cyanomethyl) pentanoic acid, a potential irreversible inhibitor of ornithine decarboxylase

(+/-)-2,5-Diamino-2-)cyanomethyl)pentanoic acid was obtained by the reaction of chloracetonitrile with the anion obtained by treatment of 3-(benzylideneamino)-2-piperidinone with sodium hydride, followed by hydrolysis in the presence of trifluoroacetic anhydride. The target compound was isolated as the monohydrochloride salt of the lactam. The compound was synthesized as a potential irreversible inhibitor of the enzyme L-ornithine decarboxylase by the mechanism generally known as suicide or Kcat inhibition. The synthesized compound produced no inhibition of the enzyme ornithine decarboxylase obtained from rat prostate gland. The inactivity of the target compound is attributed to the hydrophilicity of the cyanomethyl group.     

2-substituted 3-(aminooxy)propanamines as inhibitors of ornithine decarboxylase: synthesis and biological activity.

1-Amino-3-(aminooxy)-2-propanol (6a) has been synthesized and found to inhibit rat liver ornithine decarboxylase (ODC) with an IC50 in the nanomolar range. Compound 6a served as a basis for the design of new enzyme inhibitors, which led to the identification of 3-(aminooxy)-2-fluoropropanamine (15) as a new powerful enzyme blocker. Compound 15 inhibited ODC at 3 times lower concentrations than 6a and 3-(aminooxy)propanamine (APA), and it was superior to APA as an antiproliferative agent in inhibiting the growth of human T24 bladder carcinoma cells in vitro.     

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.     

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.     

Ontogeny of stress effects on ornithine decarboxylase activity in rats

This study demonstrates that “stress” elicits a specific pattern of organ response in developing rats that is determined by the particular “stress” and by the age of the animal. Maternal deprivation (MD) of preweanling rat pups decrease ornithine decarboxylase (ODC) activity in many tissues, as reported previously, while immobilization (IMM) and cold stress increase ODC activity in liver and heart of neonatal rat. Serum GH is decreased by MD and by IMM, but is not affected by cold stress. Stress-induced ODC elevation increases with age, while MD effects disappear at weaning. IMM and cold increase ODC activity in kidney, liver and heart of adult rats. These effects of IMM are blocked by the ganglionic antagonist chlorisondamine in adult but not in neonatal rats. The results of this study suggest that MD and the “classical” stress paradigms IMM and cold evoke different patterns of organ ODC response in neonatal rats. Furthermore, these findings suggest that the mechanism by which immobilization increases ODC activity changes from a hormonal to a neural mechanis, during ontogeny.     

Chloroform-induced multiple forms of ornithine decarboxylase: differential sensitivity of forms to enhancement by diethyl maleate and inhibition by ODC-antizyme

The role of glutathione (GSH) and ornithine decarboxylase-antizyme (ODC-AZ) in the regulation of the chloroform-mediated stimulation of rat hepatic ornithine decarboxylase (ODC) was investigated. We have previously implicated roles for each while examining the chloroform effect on crude cytosolic enzyme preparations. In this study we examined the effect of pretreatment with diethyl maleate (DEM), a GSH-depleting agent, on the chloroform stimulation of the two forms of the rat hepatic ODC enzyme and the sensitivity of these two forms to inhibition by the ODC-AZ. While the pretreatment with DEM provided a greater amount of the two forms of the ODC enzyme, it also resulted in a differential stimulation of each form when compared to chloroform alone. Additionally, Peak II was 20-25% more sensitive to the same amount of ODC-AZ then Peak I ODC activity.     

Effects of hydrazine and its derivatives on ornithine decarboxylase synthesis,activity, and inactivation

Hydrazine (1.0 mmol/kg) caused marked stimulation of rat liver ornithine decarboxylase (ODC) activity. Derivatives of hydrazine were less effective as stimulators. Enzyme activity reached a maximum about 4 hr after treatment. ODC activity in livers from dexamethasone stimulated rats was moderately inhibited when hydrazine (1.0 mmol/kg) was administered 20 min prior to sacrifice. Hydrazine also slowed inactivation halftime of ODC following dexamethasone stimulation, as measured by serial biopsy in single animals. The inactivation half-times of the treated animals fell into two populations, one responding more extensively than the other. The usefulness of a serial liver biopsy technique for determination of ODC half-time in individual animals is demonstrated.     

Ethylene glycol monomethyl ether (EGME) inhibits rat embryo ornithine decarboxylase (ODC) activity

The effects of ethylene glycol monomethyl ether (EGME) on reproductive outcome in the rat, and on ornithine decarboxylase (ODC) activity in the rat embryo were evaluated. Dams (n = 8) were treated by gavage on gestation days 6-12 (sperm = day 0) with 0, 25, 50 or 75 mg/kg EGME in 10 ml/kg distilled water. EGME had a dose-dependent effect on reproductive outcome. Gestation length was prolonged, and the number of litters delivered and neonatal body weight were reduced. Whole embryo ODC was measured on gestation days 9, 11, 13 and 15. ODC attained maximum activity in controls on day 11, increasing by more than an order of magnitude above the activity found on day 9. On day 11, a statistically significant dose-dependent inhibition of ODC activity was observed with the maximum dose of EGME inhibiting ODC activity 60 percent. On days 13 and 15, ODC activity declined markedly from peak values, and the dose-dependent inhibition was no longer evident. The study demonstrates a correlation between the inhibition of embryonic ODC activity by EGME and the effect of EGME on reproductive outcome.     

Effect of diethyl maleate on hepatic ornithine decarboxylase

Diethyl maleate (DEM), a well-known glutathione (GSH) depletor, causes a dose-dependent increase in hepatic ornithine decarboxylase (ODC) activity as well as heme oxygenase activity in rats. Considering the important role ODC has in polyamine biosynthesis in response to endogenous and exogenous stimuli, further extensive studies on the effect of DEM on ODC in relation to its GSH-depleting effect were carried out. Specifically, concomitant with the profound decrease in GSH content, the higher dose of DEM (1284 mg/kg) caused a marked increase in ODC activity (about 1000 times that of the control) at 12 hr after its administration. DEM at this dose also caused a marked increase in heme oxygenase activity, but the effects on cytochrome P-450 content and aminopyrine demethylase activity were less extensive. The increases in ODC and heme oxygenase activities evoked by DEM were almost completely blocked by pretreatment of rats with either actinomycin D or cycloheximide. Parallel to the increase in ODC activity, DEM caused a profound increase in putrescine content in the liver, while the agent reduced spermine content. The administrations of alpha-difluoromethylornithine and 1,3-diaminopropane resulted in the inhibition of DEM-mediated induction of ODC, but not heme oxygenase. In contrast, methylglyoxal bis(guanylhydrazone) inhibited the induction of both ODC and heme oxygenase evoked by DEM. The DEM-induced ODC exhibited two phases of decay with the prolonged half-lives of 26 and 223 min. Additionally, the elution profile from DEAE-Sepharose CL-6B column chromatography of cytoplasmic fraction from DEM-treated rat liver exhibited two peaks of ODC activity. These findings add new insight into the biochemical effect of DEM on hepatic polyamine metabolism in addition to its GSH-depleting effect.     

Effect of thyroxine and cortisol on brain ornithine decarboxylase activity and swimming behavior in developing rat

Thyroxine and cortisol were tested to determine their effects on the ontogenetic pattern of ornithine decarboxylase (ODC) (EC 4.1.1.17) activity in rat brain. The effects of both hormones were significant, but varied with the brain region studied and with the age of the rat. Thyroxine (1 μg/g body wt/day injected i.p. × 3) altered ODC activity in all brain regions studied, if given during the first 10 postnatal days The overall effect was to compress the time course in which ODC activity passed through its ontogenetic pattern; both the peak in activity and the drop to the characteristic low adult activity level occurred earlier and were accelerated. The magnitude of the response to thyroxine was greater in the cerebellum, followed by the cerebrum and brainstem respectively. Cortisol, when given on the first postnatal day (0.5 mg i.p. × 1), initially depressed ODC activity and subsequently prolonged the time required to reach the low level of activity found normally in adult brain. The magnitude of the response to cortisol generally was greater than that of thyroxine, except in the cerebellum. The effects of these hormones on the ontogenetic pattern of ODC activity are consistent with previously observed effects in rats both on biochemical parameters, e.g. polyamine content and nucleic acid synthesis, and behavioral parameters, e.g. development of swimming ability. Our data are consistent with the hypothesis that thyroxine and cortisol, when administered to the neonate, may exert some of their effects on nucleic acid metabolism and ultimately growth, development and behaviour via their effects on polyamine metabolism.     

Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity

Purified ornithine decarboxylase (EC 4.1.1.17, ODC) transamidated with four putrescine moieties on four glutamine residues through the action of transglutaminase (EC 2.3.2.13, TGase) purified from guinea pig liver, when added to isolated rat liver nuclei, stoichiometrically increased the activity of RNA polymerase I (EC 2.7.7.6). Te increase was relative to the pmoles of purified conjugated ODC added to the reaction and could be reinitiated after the reaction had plateaued by the further addition of ODC-putrescine conjugate. The kinetics of the reaction suggest that the ODC-putrescine conjugate was not reused but degraded after each initiation. Otherwise, the rapid plateau would not be observed. The repeated addition of 278 pmoles of purified ODC-putrescine conjugate to rat liver nuclear preparations containing 200 μg total protein consistently stimulated the incorporation of 600–700 pmoles UMP/mg protein. We suggest that ODC transmidated by its product putrescine may be the posttranslationally modified 65,000 M_r protein which has been reported by several laboratories to serve as a labile subunit of RNA polymerase I.