Hepatic Ornithine Decarboxylase Activity Research Articles

Permanent decrease in activity of ornithine decarboxylase antizyme in rat liver during chemical hepatocarcinogenesis.

This study was undertaken to determine whether or not there is failure of cellular control of L-ornithine decarboxylase activity by its antizyme, the only known natural intracellular inhibitor protein for L-ornithine decarboxylase activity, in rat liver during hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene. The formation of hepatic ornithine decarboxylase antizyme was elicited by i.p. injections of putrescine into rats fed a basal diet and rats fed the carcinogenic diet. The activities of both hepatic ornithine decarboxylase and hepatic ornithine decarboxylase antizyme were measured every month for five months, i.e., until hepatoma was fully developed. During azo-dye hepatocarcinogenesis and in fully developed hepatoma the activity of hepatic ornithine decarboxylase antizyme was always significantly lower than in normal resting liver, with minima at the second and the third months. The hepatoma does not synthesize ornithine decarboxylase antizyme more slowly than normal liver, since the difference could be neither abolished nor lessened by lengthening the time available for antizyme formation. Our results strongly suggest that the high intracellular putrescine levels in the livers of rats during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis do not exert their normal control on hepatic ornithine decarboxylase activity because of a relative inability of these preneoplastic or neoplastic cells to make the ornithine decarboxylase antizyme.

Chloroform induction of ornithine decarboxylase activity in rats.

Chloroform is a drinking water contaminant that has been demonstrated to be carcinogenic to mice and rats resulting in an increased incidence of liver and kidney tumors, respectively. The mechanism of chloroform carcinogenicity might be by tumor initiation and/or promotion. Since induction of ornithine decarboxylase (ODC) activity has been proposed as a molecular marker for tumor promoters, we have investigated the effect of chloroform on ODC activity in rats. Chloroform induced a dose-dependent increase of hepatic ODC with an apparent threshold at 100 mg/kg body weight. Female rats were two to four times more susceptible to to chloroform. Upon daily dosing of chloroform for 7 days the liver became less susceptible, with the last dose of chloroform resulting in only 10% of the activity observed after a single dose. Nuclear RNA polymerase I activity was also induced by chloroform. Chloroform, rather than increasing the activity of renal ODC, resulted in a 35% reduction. The induction by chloroform of hepatic ODC activity might be associated with regenerative hyperplasia while the renal carcinogenicity of chloroform could not be demonstrated to be associated with ODC induction.

Chloroform Induction of Ornithine Decarboxylase Activity in Rats

Chloroform is a drinking water contaminant that has been demonstrated to be carcinogenic to mice and rats resulting in an increased incidence of liver and kidney tumors, respectively. The mechanism of chloroform carcinogenicity might be by tumor initiation and/or promotion. Since induction of ornithine decarboxylase (ODC) activity has been proposed as a molecular marker for tumor promoters, we have investigated the effect of chloroform on ODC activity in rats. Chloroform induced a dose-dependent increase of hepatic ODC with an apparent threshold at 100 mg/kg body weight. Female rats were two to four times more susceptible to to chloroform. Upon daily dosing of chloroform for 7 days the liver became less susceptible, with the last dose of chloroform resulting in only 10% of the activity observed after a single dose. Nuclear RNA polymerase I activity was also induced by chloroform. Chloroform, rather than increasing the activity of renal ODC, resulted in a 35% reduction. The induction by chloroform of hepatic ODC activity might be associated with regenerative hyperplasia while the renal carcinogenicity of chloroform could not be demonstrated to be associated with ODC induction.

Alteration of hepatic ornithine decarboxylase and aniline hydroxylase activities following a single dosing of azathioprine to rats.

Hepatic ornithine decarboxylase and aniline hydroxylase activities increased as early as 6h after a single dosing of azathioprine tomale rats and reached maximum levels at the 12th h after the treatment. However, aniline hydroxylase activity dropped to levels mu lower than the cintrols 48 h following azathioprine treatment. The results are discussed with regard to the role of azqathioprine during the promotion of hepatocarcinogenesis.

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.

Ethanol-induced stimulation of hepatic ornithine decarboxylase activity in the rat.

The effect of a single feeding of ethanol on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. Ethanol (7.5 g/kg body weight) was tube‐fed to overnight‐fasted rats as a 50% (v/v) solution in water 1, 2, 3, 4, 8, 12, or 24 hr before sacrifice. The levels of ODC activity in the livers were assayed in vitro by measuring the release of 14C02 from 0L‐1‐14C‐ornithine. Hepatic ODC activities were significantly stimulated by ethanol (7.5 g/kg body weight) beginning at 1 hr and reaching a peak at 4 hr (more than a 16‐fold increase over zero time controls). Single feedings of varying doses of ethanol (2.5, 5.0, or 7.5 g/kg body weight) to overnight‐fasted rats 3 hr before sacrifice also exhibited significant increases (3 to 13‐fold) in the hepatic ODC activities. In vitro 14C‐leucir&gt;e incorporation into protein using hepatic microsomes of ethanol‐treated rats was decreased in comparison with that of controls. The ethanoMnduced stimulation of hepatic ODC activity was not abolished by pretreatment with pyrazoie, an inhibitor of ethanol metabolism. However, the stimulation of hepatic ODC activity by ethanol was suppressed by actinomycin D or cycloheximide, indicating that the enhancement is attributable to the synthesis of new enzyme protein. Furthermore, abolition of the stimulation of hepatic ODC activity due to ethanol by prior adrenalectomy suggests that the induced increase is probably mediated through stimulation of adrenal hormones. These studies demonstrate that a single dose of ethanol per os can significantly enhance in the rat the activity of hepatic ODC, a key enzyyme in the biosynthesis of polyamines, and that the effect is indirect, via adrenal hormones.

Tumor promoting phorbol-ester derivatives increase ornithine decarboxylase activity and polyamine biosynthesis in the liver of the rat and mouse.

The ability of the phorbol-ester tumor promoters to alter ornithine decarboxylase (ODC) activity in the liver of the rat and mouse was determined. The injection of 12-O-tetra-decanoyl phorbol-13-acetate (TPA, 100 microgram, i.p.) led to a 250-fold increase in hepatic ODC activity within 4 h of administration. This increase in ODC activity required both RNA and protein synthesis and did not occur when a variety of the non-tumor promoting phorbol-ester derivatives were administered to the rat. A distinct dose-dependent increase in hepatic ODC activity could be observed at 4 h following the injection of increasing amounts of TPA (0-100 microgram, i.p.). As little as 1.0 microgram TPA (i.p.) administered to a rat resulted in a significant stimulation in the activity of ODC in the liver compared to the control unstimulated values. Both 200 micrograms and 500 micrograms TPA produced less of an elevation in hepatic ODC activity than did the optimal dose of 100 micrograms. In the mouse, the administration of 1 microgram and 20 micrograms of TPA (i.p.) both led to a marked increase in hepatic ODC activity at 7 h and 4 h, respectively, following injection. A 4-5-fold increase in putrescine levels occurred in the rat liver in a biphasic manner between 4-8 h and 16-24 h following the injection of TPA (100 micrograms). No alterations in either spermidine or spermine were observed during this period. The administration of 100 micrograms of TPA to the rat did not alter the incorporated control animals. Under these identical conditions partial hepatectomy led to a large increase in DNA synthesis.

Role of pancreatic hormones in dietary induction of ornithine decarboxylase of rat liver.

1. Although the mean ornithine decarboxylase (ODC) activity induced by feeding in alloxan-diabetic rats was 60% of that in normal rats, it was markedly suppressed in pancreatectomized rats depending on the degree of pancreatectomy. 2. There was a correlation between plasma glucagon concentration and hepatic ODC activity under various dietary conditions. 3. When glucagon or insulin was given to pancreatectomized rats, dietary induction of ODC was restored to a level found in normal rats. The effects of glucagon and insulin were not additive, suggesting that these hormones act with a similar mechanism. Feeding 75% casein diet resulted in a considerable induction of ODC even in pancreatectomized rats. The enzyme was only slightly induced, however, when pancreatic hormones were given to starved rats. 4. Findings from amino acid analysis indicated that the influx of some amino acids into liver was stimulated by administration of glucagon. 5. These results suggest that increased influx of amino acids into liver is a critical factor for dietary induction of ODC and that both glucagon and insulin play a role in stimulating amino acid influx.

Induction of hepatic ornithine decarboxylase by hypolipidemic drugs with hepatic peroxisome proliferative activity.

The present investigation was conducted to determine (1) if hepatomegalic and mitogenic effects of selected hypolipidemic peroxisome proliferators are associated with an elevation of hepatic ornithine decarboxylase (ODC) levels and (2) if induction of ODC is a specific event associated with the development of preneoplastic hepatocellular foci and the eventual formation of hepatomas. Following a single i.p. dose of Wy-14,643 (Wy), the hepatic ODC activity in rats rose sharply, reaching a peak at 8 h, and returned to the normal level by 24 h. Other peroxisome proliferators tested (methyl clofenapate, BR-931 and nafenopin) also increased the hepatic ODC activities significantly 8 h after i.p.. administration of a single dose. Continuous dietary administration of Wy, whether or not these animals were previously initiated with the carcinogen diethylnitrosamine (DEN), resulted in a sustained elevation of hepatic ODC activity. Although Wy exerted an enhancing effect on DEN-initiated tumorigenesis, there was no additional increase of ODC activity. There was no correlation between the level of ODC induction and the presence or absence of altered liver cell foci. These results suggest that induction of ODC by peroxisome proliferators is not a specific event associated with the development of preneoplastic foci and hepatocarcinogenesis, but is an event associated with the sustained maintenance of hepatomegaly and and increased liver cell proliferation.

The role of putrescine in modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine.

The relationship between polyamine metabolism and DNA synthesis in regenerating liver was investigated using normal and 5-azacytidine-treated rats. The increase of DNA synthesis in regenerating rat liver was completely inhibited by administration of the analogue 1 h after partial hepatectomy. On the contrary, early and enhanced increase (modulation) of DNA synthesis was observed when the analogue was injected 24 h before partial hepatectomy. Changes in ornithine decarboxylase [EC 4.1.1.17] activity and the tissue levels of polyamines in liver remnants showed similar patterns in normal and 5-azacytidine-pretreated rats, except that the putrescine level in removed liver and the spermidine level in the S-phase were significantly higher in the drug-pretreated rats. On the other hand, a single injection of the analogue into intact rats evoked marked increase in hepatic ornithine decarboxylase activity followed by increase in the tissue level of putrescine, but not of spermidine or spermine. No similar increase in the enzyme activity was detected after injection of 5-aza-2'-deoxycytidine, which did not cause modulation of DNA synthesis. No modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine was observed when partial hepatectomy was carried out before increase of the hepatic level of putrescine. In addition, a positive and highly significant correlation was observed between the level of putrescine in the liver removed at partial hepatectomy and the extent of modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine. These results suggest that putrescine plays an important role in modulation of DNA synthesis and support our previous proposal that putrescine may have at least two roles in DNA synthesis.

Prevention of barbiturate-mediated increases of rat hepatic ornithine decarboxylase and δ-aminolevulinic acid synthetase activities by nicotinic acid and nicotinamide

Administration of secobarbital to rats produced dramatic increases in the activities of hepatic ornithine decarboxylase and δ-aminolevulinic acid synthetase at three hours which were reversed by administration of either nicotinic acid or nicotinamide. No appreciable alterations of the activity of either enzyme were observed following administration of nicotinic acid. Administration of nicotinamide produced a significant increase of ornithine decarboxylase activity, but failed to alter the activity of δ-aminolevulinic acid synthetase.

Induction of ornithine decarboxylase by biliverdin in rat liver

The administration of biliverdin (0.1mg/g of body weight) into the peritoneal cavity of rats resulted in the induction of ornithine decarboxylase in the liver. When the temporal relationships between the changes in intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level, cyclic AMP-dependent protein kinase activity and the induction of ornithine decarboxylase were investigated, the concentration of cyclic AMP increased significantly 2 h after the administration of biliverdin, while cyclic AMP-dependent protein kinase was activated after 2–4 h. The hepatic ornithine decarboxylase activity began to increase 4 h after biliverdin injection. These results suggest that there is some sequential relationship between the increase of cyclic AMP, the activation of cyclic AMP-dependent protein kinase and the induction of ornithine decarboxylase although the direct correlation of these three events remains to be elucidated.

Effect of streptozotocin-induced diabetes on hepatic ornithine decarboxylase activity in the rat.

The effect of diabetes on rat hepatic ornithine decarboxylase (ODC) activity was studied in male rats 4 h to 8 days after the ip or iv administration of streptozotocin. Hepatic ODC activity increased 4-fold above control on the fourth day of diabetes. Increased ODC activity was observed with a dose of streptozotocin (70 mg/kg iv) which increased plasma ketones and glucagon and reduced plasma insulin. No change was seen with doses causing only mild hyperglycemia without change in plasma ketones, glucagon, or insulin. Insulin therapy prevented the increase in hepatic ODC activity in diabetic rats. Adrenalectomy or hypophysectomy also prevented the diabetes-associated increase of ODC activity. The studies suggest that insulin deficiency may result in increased hepatic polyamine synthesis.

A new protocol for studying the early events during liver regeneration

Hepatic amino acid transport and ornithine decarboxylase (ODC) activity were measured as early events during liver regeneration in rats adapted to a meal-feeding protocol in which food was presented during the first 2 hr of a daily 12-hr dark period. Surgeries were performed during the early hours of the light period, and food was withheld on the day following surgery to control the effect of feeding on the parameters measured. Initial experiments indicated that rats maintained on this schedule were capable of surviving partial hepatectomy. The survival rate was greater and the fat pads larger among 56-day-old than among 42-day-old rats; this indicated that animals with greater metabolic reserves were better suited for these experiments. The hepatic α-aminoisobutyric acid (α-AIB) distribution ratio and ODC activity increased above control values by 2 hr following partial hepatectomy. The α-AIB distribution ratio peaked at 10 hr after surgery and remained above control values for an additional 16 hr. In contrast, ODC activity peaked by 4 hr after surgery, followed by two smaller peaks at 10 and 20 hr. In sham-hepatectomized rats both the α-AIB distribution ratio and the ODC activity increased above control values by 3 hr after surgery, but fell to control values by 15 hr. These findings show that increases in amino acid transport and ODC activity following partial hepatectomy occur independently of feeding. The identical time course for the initiation of both of these events suggests that they result from a common effector.

Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed.

Sequential changes in hepatic polyamine, deoxyribonucleic acid, and cyclic adenosine 3′,5′-monophosphate metabolism after subacute exposure to cadmium in rats

Heavy metal treatment (2 X 1 mg/kg per day) for 3, 5, and 7 days resulted in progressive augmentation in the incorporation of [14C]thymidine into hepatic DNA. In contrast with the observed enhancement in DNA synthesis, cadmium exposure tended to produce a decrease in the activity of hepatic ornithine decarboxylase (EC 4.1.1.17) at 1, 3, or 5 days with the lowest (34% of control values) enzymic activity seen after 7 days. A similar reduction in the activity of S-adenosylmethionine decarboxylase (EC 4.1.1.50) was observed in livers of rats treated with cadmium for 1-7 days. Subacute exposure to cadmium significantly lowered the hepatic levels of spermidine and spermine whereas the endogenous concentrated of putrescine remained unaltered. In addition to the observed effects on the biosynthesis of polyamines and DNA, heavy metal treatment produced stimulation of the hepatic adenylate cyclase (EC 4.6.1.1)--cyclic AMP system. Significant increases in the activity of hepatic adenylate cyclase and endogenous cyclic AMP levels were detected as early as 1 day and the observed alterations persisted during the entire 1-week period of cadmium exposure. The depression in polyamine formation was accompanied by enhanced DNA biosynthesis as well as stimulation in the adenylate cyclase-cyclic AMP system of rat liver.

Tissue-specific stimulation of ornithine decarboxylase activity by pituitary factors immunologically related to growth hormone.

Ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) is an important enzyme in polyamine synthesis. Its activity is influenced by several peptides hormones, including growth hormones, which have physiological significance in various growth situations. A crude ovine pituitary growth hormone preparation (NIH-GH-S10) was subjected to gel exclusion chromatography (Sephadex G-100) and two major fractions were obtained. One of these corresponded to dimeric growth hormone (GH). The other fraction was excluded by the gel matrix, suggesting a material of higher molecular weight than GH. This was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of a high molecular weight fraction by radioimmunoassay (antisera prepared against GH) and by bioassay (weight gain in hypophysectomized rats) gave apparent GH contents of 19% and 6%, respectively. On a weight basis, the high molecular weight fraction was more effective than GH in stimulating the activity of hepatic and adrenal ornithine decarboxylase, but GH was more effective in stimulating renal ornithine decarboxylase activity. Subfractionation of the high molecular weight fraction using a high porosity gel (Sephadex G-200) gave four fractions, which were shown by amino acid analysis and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to be distinct from GH and heterogenous. These subfractions had different potencies for stimulating renal and hepatic ornithine decarboxylase activity. The ability of crude growth hormone preparations to stimulate ornithine decarboxylase activity in some tissues may be a function of pituitary factors, in addition to GH, which have minimal growth promoting activity.

Is Cyclic Amp the Regulator of Hepatic Ornithine Decarboxylase Activity?

The role of cyclic AMP in the regulation of hepatic ornithine decarboxylase (ODC) activity in the rat was studied in the whole animal and in the perfused organ. Dibutyryl cyclic AMP or butyrate given to intact rats increased ODC activity; this increase was abolished by hypophysectomy 1 h prior to administering ether compound. Administration of 1 mg 1-methyl-3-isobutylxanthine (MIX) to intact rats increased ODC activity within 4 hours whereas hypophysectomy 1 h before treatment prevented this increase. No change in hepatic cyclic AMP content was seen in either intact or hypophysectomized rats following MIX. Perfusion with 0.5 mM dibutyryl cyclic AMP decreased ODC activity in isolated livers whereas perfusion with 0.5 mM 8-bromocyclic GMP produced a small increase in ODC activity. These data suggest that the effect of dibutyryl cyclic AMP in intact animals may be a property of the butyrate and that this action as well as the action of MIX may be mediated through the permissive effect of pituitary and/or adrenal hormones. The normal hepatocyte does not increase its ornithine decarboxylase activity after direct exposure to dibutyryl cyclic AMP.

Evidence for structural dissociation of two biologic actions of growth hormone

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase ( l-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6–124 and 150–179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125–149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observation indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.

Stimulation of ornithine decarboxylase and nuclear RNA polymerase activity in rat liver by glucagon and dibutyryl cyclic amp.

ABSTRACT The influence of glucagon and dibutyryl cyclic 3′,5′-AMP on ornithine decarboxylase (EC 4.1.1.17) and nuclear RNA polymerase (EC 2.7.7.6) activity of rat liver was studied. Intraperitoneal administration of 0.2 mg of glucagon per 100 g body weight resulted in a four- to six-fold increase in ornithine decarboxylase activity. This enzyme activity was also markedly increased by a single ip injection containing 2.5–5 mg of dibutyryl cyclic AMP. The time course of this effect demonstrated a maximum at 3–4 h. Both agents also stimulated nuclear RNA polymerase activity. The increase in hepatic ornithine decarboxylase activity by dibutyryl cyclic AMP was not abolished by adrenalectomy but was totally prevented by the administration of actinomycin D, α-amanitin and cycloheximide, which suggests that the increase in activity represents synthesis de novo of the enzyme. A sharp rise in liver ornithine decarboxylase activity was also observed after administration of theophylline, whereas no change in enzyme activity was found after the administration of adenosine or adenosine 5′-nucleotides.