Effect of carbon tetrachloride on polyamine metabolism in rodent liver

Administration of hepatotoxic doses of carbon tetrachloride to mice produced a 25-fold increase in spermidine/spermine N¹-acetyltransferase activity within 6 h, but did not significantly change the activity of polyamine oxidase. The content of acetylated polyamines in the mouse liver was increased more than 100-fold from levels below the limit of detection to 0.6 μmol of N¹-acetylspermidine and 0.045 μmol of N¹-acetylspermine per gram of tissue. Putrescine levels also rose by 7-fold within 6 h and by 21-fold within 24 h. These results are in contrast to changes in hepatic polyamines brought about in the rat by carbon tetrachloride. Although the hepatotoxin produced a similar increase in spermidine/spermine N¹-acetyltransferase in this species, the rise in acetylated polyamines was much smaller and more transient. The content of N¹-acetylspermidine was increased only to 0.066 μmol/g and N¹-acetylspermine was not detected. However, in the rat putrescine increased 35-fold within 6 h and 64-fold by 16 h. These differences appear to be due to the much higher polyamine oxidase activity which was 20 times greater in the rat than in the mouse liver. This oxidase converts N¹-acetylspermine to spermidine and degrades N¹-acetylspermidine to putrescine. Spermine content was significantly reduced in both species after exposure to carbon tetrachloride, but only part of this decline could be attributed to the increased acetylation.     

Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride into rats resulted within 12 hours in a marked accumulation of putrescine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the maximal accumulation of putrescine there was a marked incorporation of radioactivity from labelled spermidine to liver putrescine $\text{in vivo}$. The conversion of spermidine to liver putrescine was hardly detectable in control animals. Besides the treatment with carbon tetrachloride, increased conversion of radioactive spermidine to liver putrescine $\text{in vivo}$ also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, $\text{i. e.}$ under circumstances characterized by a stimulation of ornithine decarboxylase activity and an increased accumulation of putrescine.     

Comparison of polyamine and S-adenosylmethionine contents of growing and encysted Acanthamoeba isolates

We have used High Performance Liquid Chromatography to determine metabolite characteristics of three recent isolates of Acanthamoeba which exhibit cultural characteristics consistent with those of established potential pathogens. Growing amoebae and dormant cysts of these isolates were explored in regard to their qualitative and quantitative intracellular levels of polyamine and S-adenosylmethionine metabolites. The polyamine found in the greatest concentration in the growing cells was 1,3-diaminopropane (DAP), followed by spermidine (SPD). A low level of putrescine was also found in the growing cells. These polyamines significantly decreased in concentration as the amoebae differentiated to cysts. N⁸-acetylspermidine and acetylspermine were found in both developmental stages while acetylcadaverine was found only in growing amoebae and N¹-acetylspermidine only in cysts. Acetylputrescine was present in both stages of two isolates but only in the growing amoebae of the third isolate. Spermine was not detected in any of the isolates.S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were present in growing amoebae but SAM was undetectable or barely detectable in cysts. SAH also decreased in concentration during encystation of two of the isolates to a level comparable to that of the other isolate.The developmental transition from growing amoebae to dormant cysts is characterized metabolically by a threshold adjustment in concentration of SAM, SAH and of the polyamines (esp., DAP and SPD).     

Deacetylation of N8-acetylspermidine by subcellular fractions of rat tissue

The in vitro deacetylation of N⁸-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N¹-acetylspermidine differed from that observed for N⁸-acetylspermidine and does not appear to involve a simple deacetylation reaction.     

Determination of the naturally occurring monoacetyl derivatives of di- and polyamines

A method is described for the determination of pmol quantities of monoacetylputrescine, N¹-acetylspermidine, N⁸-acetylspermidine and related compounds. The method is based on the derivatization of these compounds with 5-dimethylaminonaphthalene-1-sulphonyl-chloride, followed by thin-layer chromatographic separation. Cleanup steps allow the application of the method to urine analyses. From the repeated determination of acetylated polyamines in the urine of healthy individuals it can be concluded that these conjugates are the major excretory form of di- and polyamines.The cleanup steps used in this procedure and the method described for the stabilization of 5-dimethylaminonaphthalene-1-sulphonyl derivatives on thin-layer plates are advantageous also for the analyses of total polyamines in urine hydrolysates, and in related applications of the dansylation method.     

A new enzyme-linked immunosorbent assay (ELISA) for studying immunocytochemical procedures using an antiserum produced against spermidine as a model

Antiserum was produced in rabbits against the polyamine spermidine (Spd) conjugated to bovine serum albumin (BSA). The reactivity of the serum to Spd and a variety of structurally related compounds was quantified by a new immunocytochemical model system incorporating an enzyme-linked immunosorbent assay (ELISA) binding test. This is based on the principle of coupling these compounds to the wells of microtiter plate activated with poly-l-lysine and glutaraldehyde and incubating the wells by the indirect immunoperoxidase method. The antiserum showed a 25% cross reaction with spermine (Spm), putrescine (Put), and cadaverine (Cad), and a 1% cross reaction with 1,3-diaminopropane (Dap), but no cross reaction with monoacetyl polyamines and amino acids. The antibody binding was inhibited most effectively by absorption of the antiserum with N ¹-acetylspermidine and Spd in the ELISA inhibition test. Also, immunoblot analysis of the antiserum with nitrocellulose paper gave completely identical results to the ELISA binding tests. Spd-like immunoreactivities in human melanoma BD and neuroblastoma IMR 32 cell lines are presented as examples of the staining pattern obtained with the antiserum. Absorption of the serum with N ¹-acetylspermidine and Spd was demonstrated to abolish the immunostaining reaction. The immunohistochemical model is simple: amines and amino acids are bound in the same way as in aldehyde-fixed tissues and, in comparison to immunoblot analysis, the immunoreactivity can be more easily and accurately quantified by assay with the antibody. The model should prove useful in assessing the specificity of other antisera.     

Occurrence in high concentrations of N1-acetylspermidine and sym-homospermidine in the hamster epididymis

In mature hamster epididymis several unknown peaks were observed on our high-performance liquid chromatograms in addition to the common polyamines, putrescine, spermidine and spermine. Three of the peaks were identified as N¹-acetylspermidine, N¹-acetylspermine and sym-homospermidine by means of thin-layer chromatography, gas chromatography-mass spectrometry and acid hydrolysis. The concentrations of N¹-acetylspermidine and sym-homospermidine were highest in the distal caput epididymidis among epididymal regions studied. This is the first report to show that sym-homospermidine occurs in mammalian tissues.     

The determination of N1-acetylspermine in mouse liver

N¹-Acetylspermine has been postulated to be an intermediate in the conversion of spermine to spermidine. This compound, together with N¹-acetylspermidine has now been detected in the liver of mice which were pretreated with tetrachloromethane. The following methods were used for the identification of N¹-acetylspermine: (a) High-pressure liquid-chromatography of the non-derivatized amines on a reversed-phase column, using octane sulfonate for ion-pairing. (b) Thin-layer chromatography of the dansyl derivatives. (c) Mass spectrometry of the dansyl derivatives. Both chromatographic methods allowed the quantitative estimation of N¹-acetylspermine and N¹-acetylspermidine in the liver of tetrachloromethane-treated animals.     

Acetylation of spermidine in polyamine catabolism

Treatment with thioacetamide (150 mg/kg)_ was used to enhance polyamine metabolism in rat liver. The increased uptake and catabolism of [¹⁴C]spermine and the changes of putrescine, spermidine and spermine concentrations indicated enhanced polyamine turnover rates. The increase of hepatic putrescine concentration was accompanied by an increase of monoacetylputrescine and N¹-monoacetylspermidine concentration. In control animals, the latter compound was below detection levels. Thioacetamide treatment also enhanced putrescine excretion, which again was concomitant with an increased excretion of N¹-acetylspermidine.The close time-dependent correlation between induced putrescine formation and enhanced formation of N¹-acetylsperimidine at a time when liver spermidine and spermine concentrations are not changed, favors the notion that acetylation is an essential step in polyamine degradation and elimination. The increase of polyamine oxidase and decrease of acetylpolyamine deacetylase activities in the liver of thioacetamide-treated rats is in line with an increased polyamine turnover, but these enzymes. although essential, are not rate-limiting in the catabolic reactions.     

The effect of diet on carbon tetrachloride metabolism

1. Blood and liver concentrations of carbon tetrachloride were measured, at intervals after an oral dose, in rats given stock and protein-free diets. The values did not correlate with the resistance to poisoning found in the rats on protein-free diets. 2. The metabolism of carbon tetrachloride to carbon dioxide in vivo and in liver microsomal preparations was depressed in animals given protein-free diets. 3. Rats given a single dose of DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] were highly sensitive to carbon tetrachloride poisoning. The livers of such animals had an increased microsomal protein content and greatly increased microsomal activity in the demethylation of Pyramidon (aminopyrine) and in the conversion of ¹⁴CCl₄ into ¹⁴CO₂. 4. The incorporation of [¹⁴C]leucine into protein by liver slices was depressed by carbon tetrachloride. This effect was decreased by addition of SKF525A (2-diethylaminoethyl 2,2-diphenyl-2-propylacetate) and in slices from rats given protein-free diets. It is suggested that the toxicity of carbon tetrachloride is closely linked to its metabolism.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.     

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N⁴-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N⁴-bis(aminopropyl)-N¹-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N⁴-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.     

The effect of carbon tetrachloride on lysosome function in kidneys and livers of mice

Lysosomal function in the degradation of intravenously injected ¹²⁵I-labelled albumin was evaluated in carbon tetrachloride-injected and control mice. A transient effect was noted in the kidneys at 1 h after injection of the solvent, but at later times the effects on phagolysosome formation and function were minimal and were similar in liver and kidneys. The results suggest that lysosomes are not involved in the hepatotoxicity of carbon tetrachloride.     

Conversion of exogenous spermidine into putrescine after administration to rats

Administration of large, but non-toxic doses of spermidine (0.4–1.25 mmol/kg) led to a substantial increase in putrescine in liver, kidney and a number of other tissues including muscle. The increase in putriscine peaked at 6 h after treatment and was completely prevented by administration of cycloheximide 3 h after the spermidine suggesting that the induction of a new protein was required. This protein is likely to be spermidine N¹-acetyltransferase which was induced by the treatment with spermidine and increased 3–4-fold in liver and kidney within 6 h. N¹-Acetylspermidine was detected in tissues at this time after spermidine treatment and experiments in which labeled spermidine was given indicated that a substantial fraction of the administered spermidine was converted into N¹-acetylspermidine and into putrescine. These results suggest that the rise in putrescine after spermidine treatment is brought about by the production of N¹-acetylspermidine which is converted into putrescine by the action of polyamine oxidase. The limiting step in this conversion is the activity of the acetylase which is induced in response to the rise in spermidine content. The acetylase/oxidase pathway, therefore, provides a means by which polyamine levels can be regulated and excess polyamine disposed of.     

Determination of monoacetyldiamines and polyamines in urine by high-performance liquid chromatography

A procedure is described for the determination of monoacetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine in human urine. The procedure is based on the high-performance liquid chromatographic separation of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivatives of these amines using two different chromatographic modes. Monoacetyl-1,6-diaminohexane was used as an internal standard. The amines were extracted from urine using a silica gel cartridge. The dansyl monoacetylpolyamines were separated from the mixture of dansyl derivatives of urinary amines on a bonded-phase CN column using a programmed solvent gradient elution. The dansyl acetylpolyamines were rechromatographed on a silica gel column.This chromatographic procedure was used for the determination of the concentration of N¹-acetylspermidine, N⁸-acetylspermidine and monoacetylputrescine in the urine of healthy volunteers and cancer patients.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

Acetylated polyamines as substrates for human pregnancy serum diamine oxidase

Human pregnancy serum diamine oxidase was purified 50 fold and tested for activity with a variety of substrates. Putrescine, spermidine, spermine, N-acetylputrescine, N⁸-acetylspermidine, and N¹-acetylspermidine were acceptable substrates for the enzyme, which exhibited greatest activity against N¹-acetylspermidine.     

A selective inhibitor of N8-acetylspermidine deacetylation in mice and HeLa cells without effects on histone deacetylation

The inhibitory effects of 7-[N-(3-aminopropyl)amino]heptan-2-one (APAH) on N8-acetylspermidine deacetylation were studied. In in vitro studies, APAH produced inhibition (apparent Ki of 0.18 microM) of N8-acetylspermidine deacetylation by the 100,000g supernatant fraction of rat liver. This apparent Ki was 60-fold less than the apparent Km (11 microM) for deacetylation of the substrate, N8-acetylspermidine, suggesting that APAH could be a potent, effective inhibitor in vivo. APAH was administered to mice by intraperitoneal injection at a dose of 200 mg/kg, and polyamine and acetylpolyamine levels in liver and spleen were measured. In tissues of control mice, N8-acetylspermidine was not detectable but increased to detectable levels 30-360 min after APAH treatment. These data are consistent with inhibition of the deacetylase by APAH. Increases in putrescine and N1-acetylspermidine levels occurred in liver after APAH treatment with increases in N1-acetylspermidine levels observed in spleen. In HeLa cells, a significant increase in N8-acetylspermidine was observed following 24 h exposure to 10 microM APAH while no change occurred in the acetylation level of HeLa cell histones. In contrast, 24 h exposure to 10 mM sodium butyrate produced no change in N8-acetylspermidine levels and an increase in the acetylation level of histones H4 and H2B. These results suggest that APAH has a relatively selective inhibitory effect on N8-acetylspermidine but not histone deacetylation. This is the first report of significant levels of N8-acetylspermidine in animal tissues and of the effects of in vivo inhibition of N8-acetylspermidine deacetylase.     

The ability of salts to inhibit the reaction between periodate anions and ovotransferrin.

N1-Acetylspermidine is not detectable in rat heart, but its content greatly increases after a single injection of isoprenaline (10 mg/kg), reaching a concentration of about 10 nmol/g of tissue 4 h after the treatment. Part of the accumulated N1-acetylspermidine was split to putrescine. Isoprenaline also caused an increase of N1-acetylspermidine in the spleen, where its concentration increased 3.5-fold 6 h after the catecholamine. The accumulation of N1-acetylspermidine was dependent on the dose of isoprenaline in both the heart and the spleen, and was strongly inhibited by beta-antagonists and inhibitors of protein synthesis.     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine