Effects of phorone (diisopropylidene acetone), a glutathione (GSH) depletor, on hepatic enzymes involved in drug and heme metabolism in rats: evidence that phorone is a potent inducer of heme oxygenase

Concomitant with the depletion of glutathione content, phorone (250 mg/kg, ip.) produced a marked increase in heme oxygenase activity, biphasic effect on delta-aminolevulinic acid synthetase activity, and slight decreases in cytochrome P-450 content and aminopyrine demethylase activity in the liver of rats. The increase in heme oxygenase activity evoked by phorone was almost completely blocked by pretreatment of rats with actinomycin D and cycloheximide. Phorone was able to produce the changes in these parameters in a dose-dependent manner. Buthionine sulfoximine, a GSH depletor by inhibition of biosynthesis, failed to affect these hepatic parameters.     

Induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase by sulfobromophthalein in rats

The administration of sulfobromophthalein (BSP, 0.5 mmol/kg, ip.) increased ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities to 30-fold and 5-fold, respectively, of the controls at 12 hr in the liver of rats. Parallel to the increase in ODC, there was an increase in hepatic putrescine content. However, spermine content tended to decrease. BSP increased ODC and SAMDC activities and putrescine content, but decreased spermine content, in a dose-dependent manner. Pretreatment of rats with actinomycin D and cycloheximide almost completely blocked the BSP-mediated increase of ODC and SAMDC activities. Pretreatment with glutathione (GSH) failed to inhibit BSP-mediated increase of ODC and SAMDC activities. In addition, the administration of BSP-GSH conjugate (0.5 mmol/kg, iv.) did not produce the increase of ODC and SAMDC activities. Pretreatment with phenobarbital and 3-methylcholanthrene did not inhibit BSP-mediated increase of ODC and SAMDC. The results indicate that BSP could cause changes in hepatic polyamine content due to the induction of ODC and SAMDC.     

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats

Trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. Cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. Trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.     

Effects of phorone and/or buthionine sulfoximine on teratogenicity of 5-fluorouracil in mice

Embryotoxicity and teratogenicity of 5-fluorouracil (5-FU) and modulation of its effect by the depletors of glutathione (GSH) were evaluated in mice. Pregnant ICR mice were intraperitoneally (i.p.) injected with 25 mg/kg of 5-FU on day 11 of gestation (vaginal plug = day 0). Mice were pretreated i.p. with 250 mg/kg of phorone, a GSH depleting agent and/or 200 mg/kg of buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis) 4 hours before dosing with 5-FU. Dams were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations. Pretreatment with phorone or BSO decreased fetal weight and increased the frequency and severity of oligodactyly induced by 5-FU, as well as the reduction of maternal GSH levels. Combined use of 125 mg/kg phorone and 100 mg/kg BSO i.p. augmented growth retardation induced with 5-FU. Cotreatment with exogenous GSH, at a dose of 300 mg/kg injected intravenously, could not suppress the augmentative effects of phorone and/or BSO on 5-FU teratogenicity under these experimental conditions. These results indicate that the level of endogenous GSH is one of the factors which significantly affects teratogenicity of 5-FU.     

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.     

Nitric Oxide Synthase Inhibition Promotes Carcinogen-Induced Preneoplastic Changes in the Colon of Rats

l-Arginine is metabolized either to polyamines through arginase and ornithine decarboxylase (ODC) activities or to citrulline and nitric oxide (NO, nitrogen monoxide) through the NO synthase (NOS) pathway. Polyamine levels and ODC activity are high in tumor cells. The aim of this study was to test whether N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NOS, modulates colon carcinogenesis. Adult male Wistar rats were treated with azoxymethane (AOM, 15 mg/kg ip), a chemical carcinogen, once a week for 2 weeks. One week after the second injection the rats were randomly divided into two groups. One group (n = 8) received l-NAME (10 mg/kg body wt/day) in drinking water. The control group (n = 8) received tap water. After 5 weeks, the rats receiving l-NAME showed enhanced mean basal arterial blood pressure, decreased heart rate, and a significant decrease of the cGMP content in the colonic mucosa. In both groups, AOM induced the formation of colonic aberrant crypt foci (ACF). In l-NAME-treated rats, the number of ACF was higher than in controls by 47%. ODC activity was enhanced by 11-fold. S-Adenosyl-methionine-decarboxylase activity and putrescine concentration were significantly increased in the colonic mucosa of l-NAME-treated rats. The data suggest that l-NAME promotes carcinogen-induced preneoplastic changes in the colon by inhibiting NOS activity and by stimulating polyamine biosynthesis.     

Induction of hepatic and renal ornithine decarboxylase by cobalt and other metal ions in rats.

We previously showed that Cd2+ is able to induce hepatic and renal ornithine decarboxylase (ODC). In addition to Cd2+, the administration of Co2+ and other metal ions such as Se2+, Zn2+ and Cr2+ produced a significant increase of hepatic and/or renal ODC activity. Of the metal ions used in this study, Co2+ produced the greatest increase of ODC activity. The maximum increases in hepatic and renal ODC activity, to respectively 70 and 14 times the control values in male rats, were observed 6 h after the administration of Co2+. A similar response was seen in the liver, but not in the kidney, of female rats. Thereafter, ODC activity gradually returned to control values in the liver, but it was profoundly decreased to 7% of the control value at 24 h in the kidney. The pretreatment of animals with either actinomycin D or cycloheximide almost completely blocked the Co2+-mediated increase of ODC activity. Co2+ complexed with either cysteine or glutathione (GSH) failed to induce ODC. Depletion of hepatic GSH content by treatment of rats with diethyl maleate greatly enhanced the inducing effect of Co2+ on ODC. The inhibitors of ODC, 1,3-diaminopropane and alpha-difluoromethylornithine, were able to inhibit the induction of the enzyme, without affecting the induction of haem oxygenase by Co2+. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, significantly inhibited the Co2+-mediated induction of both ODC and haem oxygenase. It is suggested that the inducing effects of Co2+ on ODC and haem oxygenase are brought about in a similar manner.     

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.     

Dietary induction of ornithine decarboxylase in male mouse kidney

In male mouse kidney, ornithine decarboxylase (ODC) is induced after feeding, and the induction depends on dietary protein content. 24 h after feeding with 50% casein-containing meal, ODC activity and amount of immunoreactive ODC protein increased more than 10-fold, ODC mRNA level increased 2-fold, and the ODC half-life extended 7-fold. The renal ODC induction after feeding is, therefore, due mainly to stabilization of ODC protein. Urinary excretion of putrescine increased in response to the ODC induction, but the renal polyamine contents scarcely changed. Consistently, the level of antizyme, a polyamine-inducible protein, determined as the ODC-antizyme complex level, scarcely changed after feeding, and the antizyme/ODC ratio in the kidney largely decreased, resulting in the stabilization of ODC protein. The present results suggest that the strong excretion system of the kidney for newly synthesized polyamines enables renal ODC escape from antizyme-mediated feedback regulation.     

Ovarian function in the rat following irreversible inhibition of L-ornithine decarboxylase

The possibility that the transient rise in rat ovarian ornithine decarboxylase (ODC) activity and the associated increase in putrescine which occur under luteinizing hormone control late in proestrus have an essential role in ovarian function has been tested using DL-α-difluoromethylornithine (DFMO) an irreversible inhibitor of ODC. Treatment with DFMO, 500 mg/kg s.c. at 12:00 h on the day of proestrus and 200 mg/kg, 6, 12 and 18 h thereafter completely suppressed both the rise in ovarian ODC activity and the associated increase in putrescine concentrations. However, ovulation took place normally under these conditions and the course of the resulting pregnancies was also normal. Similarly, combined treatment with DFMO and an inhibitor of S-adenosyl-L-methionine decarboxylase, 1,1-((methylethanediylidine)-dinitrilo) bis (3-aminoguanidine), 25 mg/kg, given at 10:00 h on the morning of proestrus failed to influence either ovulation or the subsequent period of gestation. These data provide no support for a functional role of the pre-ovulatory rise of ODC in rat ovary in the major peri-ovulatory events of that particular cycle, although they do not exclude effects on systems (e.g. steroidogenesis) not directly examined with our experimental approach. On the other hand, inhibition of ODC resulted in an increase in the number of uterine implantation sites following mating at the next proestrusestrus 4 days later. These data would support the previously expressed view that ODC may be associated with the gonadotrophin-induced initiation of follicular development for ovulation in the succeeding cycle. Moreover, since inhibition of the enzyme resulted in facilitation of the process, the normal physiological function of ODC, and/or the putrescine generated through its action, would appear to be inhibitory.     

Effect of stress and sympathetic activity on rat cardiac and aortic ornithine decarboxylase activity.

Adult male rats were injected either with α- or ß-adrenergic agonists and/ or antagonists and ornithine decarboxylase (ODC) activity in the heart and aorta was measured 4 hours later. At the lower doses, isoproterenol (0.2–0.4 mg/kg) resulted in a 10-fold increase in cardiac ODC activity but caused no significant change in aortic ODC activity. In contrast, phenylephrine (1 mg/kg) caused a 4-fold increase in aorta but no change in cardiac ODC activity levels. Phenoxybenzamine pretreatment completely abolished the PE-induced increase whereas no change was seen in ISop injected animals. Similarly, pretreatment with propranolol blocked the ISop induced response on ODC activity but had no effect on the increases observed after PE. These data suggest that the sympathetic regulation of ODC activity levels is mediated primarily via the ß-receptor in the heart but through the α-receptor in the aorta.     

Thyroid function and polyamines. III. Changes in ornithine decarboxylase activity and polyamine contents in the rat thyroid during hyperplasia and involution

Changes in ornithine decarboxylase (ODC) activity and in polyamine contents of the rat thyroid were studied under various experimental conditions. Methylthiouracil (MTU) treatment produced several-fold increases in the thyroid ODC activity and in the content of putrescine, spermidine and spermine within a week. While serum thyrotropin (TSH) levels increased gradually up to 3 weeks, the content of both putrescine and spermidine tended to reach a plateau after 2 weeks of the goitrogen treatment; spermine content continued to increase progressively for 3 weeks. Discontinuance of MTU at 7 days resulted in a rapid decline in the elevated thyroid ODC activity, followed by a diminution of putrescine, spermidine and RNA contents. Thyroidal putrescine, spermidine and RNA responded more sensitively to both introduction and withdrawal of TSH stimulation than thyroidal spermine and DNA. Excess iodide, having no effect on the basal level of thyroid ODC, suppressed the MTU-induced increase in this enzyme activity without affecting circulating TSH, thyroxine (T4) and triiodothyronine (T3) levels. There was a significant negative correlation between the ODC activity and intrathyroidal concentration of iodine in MTU-pretreated rats. Theophylline increased the thyroid weight and ODC activity when given to rats fed with a subeffective dose of MTU. Analyses of serum TSH, T4, T3 and of thyroidal iodine revealed that TSH-induced thyroid ODC activity was suppressed by increased circulating thyroid hormones and/or intrathyroidal iodine. Furthermore, it was suggested that thyroid hormones and excess iodide acted directly on the thyroid to alter polyamine biosynthesis, possibly by changing the responsiveness of the gland to TSH.     

Anti-hypoxic effect of glutathione depletors

The effect of various reduced glutathione (GSH) depletors on the survival time under normobaric and hypobaric hypoxia was examined in mice. The survival time was markedly prolonged in mice treated with glutathione S-transferase substrate, 2-cyclohexene-1-one (50-100 mg/kg, ip) and phorone (100-250 mg/kg, ip). The anti-hypoxic effect lasted for at least 3 hr and the maximum effect was found 0.5 hr after injection. Further, both compounds significantly elevated blood glucose levels 0.5-1 hr after treatment. The extent of the elevated blood glucose was nearly comparable to that of the mice treated with glucose (1-2 g/kg, ip), which was found to possess an anti-hypoxic effect. However, a GSH synthesis inhibitor, buthionine sulfoximine, could cause neither a prolongation of survival time of hypoxic mice nor an elevation of blood glucose. Moreover, unlike the depletion of hepatic GSH, brain GSH was markedly decreased by 2-cyclohexene-1-one and phorone, but not by buthionine sulfoximine. These findings suggest that the elevated blood glucose may involve in one of the mechanisms of the anti-hypoxic effect of 2-cyclohexene-1-one and phorone. A relationship between the anti-hypoxic effect and the depletion of brain GSH was also discussed.     

A stable, inducible, dose-responsive ODC overexpression system in human cell lines

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.     

Glyceryl trinitrate,a nitric oxide donor,abrogates ferric nitrilotriacetate-induced oxidative stress and renal damage

Ferric nitrilotriacetate (Fe-NTA), a common water pollutant and a known renal carcinogen, acts through the generation of oxidative stress and hyperproliferative response. In the present study, we show that the nitric oxide (NO) generated by the administration of glyceryl trinitrate (GTN) affords protection against Fe-NTA-induced oxidative stress and proliferative response. Administration of Fe-NTA resulted in a significant (P<0.001) depletion of renal glutathione (GSH) content with concomitant increase in lipid peroxidation and elevated tissue damage marker release in serum. Parallel to these changes, Fe-NTA also caused down-regulation of GSH metabolizing enzymes including glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase and several fold induction in ornithine decarboxylase (ODC) activity and rate of DNA synthesis. Subsequent exogenous administration of GTN at doses of 3 and 6mg/kg body weight resulted in significant (P<0.001) recovery of GSH metabolizing enzymes and amelioration of tissue GSH content, in a dose-dependent manner. GTN administration also inhibited malondialdehyde (MDA) formation, induction of ODC activity, enhanced rate of DNA synthesis, and pathological deterioration in a dose-dependent fashion. Further, administration of NO inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), exacerbated Fe-NTA-induced oxidative tissue injury, hyperproliferative response, and pathological damage. Overall, the study suggests that NO administration subsequent to Fe-NTA affords protection against ROS-mediated damage induced by Fe-NTA.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Enhancement of murine phenytoin teratogenicity by the gamma-glutamylcysteine synthetase inhibitor L-buthionine-(S,R)-sulfoximine and by the glutathione depletor diethyl maleate

The teratogenicity of phenytoin may result from its enzymatic bioactivation to a reactive intermediate, which, if not detoxified, can interact with embryonic tissues and alter development. Glutathione (GSH) is an important cofactor/substrate for many physiological processes and for the detoxification of xenobiotic reactive intermediates. This study examined the effects of the GSH depletor diethyl maleate (DEM) and the GSH synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO) on phenytoin embryopathy. Phenytoin, 55 mg/kg, was administered intraperitoneally (ip) to pregnant CD-1 mice at 0900 hr on gestational days 12 and 13. Pretreatment with DEM, 150 or 300 mg/kg ip, enhanced the incidence of phenytoin-induced cleft palates by 3.3-fold and 2.3-fold, respectively (P less than 0.05), without affecting the incidence of resorptions, postpartum death, or mean fetal weight. BSO, 1,800 mg/kg ip, given 0.5 hr prior to phenytoin, resulted in a 2.4-fold increase in postpartum lethality and a 5-fold increase in fetal weight loss (P less than 0.05), without altering the incidence of resorptions or cleft palates. In two subsequent studies, BSO, 680-1,018 mg/kg/day, was given in the drinking water on gestational days 9 to 13 in the first study and on days 10 to 14 in the second study. Phenytoin, 55 mg/kg ip, was given on days 11 and 12 and on days 11 to 13 in the respective studies. In the first drinking water study, BSO enhanced the incidence of phenytoin-induced fetal resorptions 3.8-fold and cleft palates 3.3-fold (P less than 0.05) but did not affect postpartum death. In the second study, BSO enhanced the incidence of resorptions, cleft palates, and postpartum death by 2-fold, 2.6-fold, and 1.7-fold, respectively (P less than 0.05). In both of the latter two studies, phenytoin-induced fetal weight loss was altered by BSO treatment (P less than 0.05). BSO alone had no embryopathic effects. These results suggest that GSH may be involved in the detoxification of a reactive intermediate of phenytoin and/or in fetal cytoprotection.     

Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.     

Induction of Ornithine Decarboxylase in Cerebral Cortex by Excitotoxin Lesion of Nucleus Basalis: Association with Postsynaptic Responsiveness and N-Methyl-d-Aspartate Receptor Activation

The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)