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Effect of carbon tetrachloride on polyamine metabolism in rodent liver
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol.
217, No. 2, September,
pp. 730-737,
1982
Effect of Carbon Tetrachloride on Polyamine Metabolism in Rodent Liver’ HANNU
P&is
ANTHONY
AND
E. PEGG’
Departmmzt of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, Pennsylvania State University, 500 University Drive, Hershey, Pennsylvania 17088 Received
April
30, 1982
Administration of hepatotoxic doses of carbon tetrachloride to mice produced a 25fold increase in spermidine/spermine Nr-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 loo-fold from levels below the limit of detection to 0.6 pmol of N1-acetylspermidine and 0.045 pmol 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 N’-acetyltransferase in this species, the rise in acetylated polyamines was much smaller and more transient. The content of N’-acetylspermidine was increased only to 0.066 pmol/g and N’-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 N’-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.
Polyamine metabolism in rat liver is dramatically affected by exposure to the hepatotoxin, carbon tetrachloride (l-3). Such exposure leads to the induction of an acetylase which acetylates spermidine and spermine at the N’ position (2, 3). Although this enzyme increases manyfold within a few hours, only small amounts of N’-acetylspermidine were detected in the liver at times corresponding to the peak of acetylase activity and monoace-
tylspermine was not detected at all (3-5). It was suggested (3,5) that the absence of large quantities of these acetylated derivates was due to their rapid oxidation by the enzyme polyamine oxidase which converts monoacetylspermine to spermidine and N’-acetylspermidine into putrescine (6, 7). The striking accumulation of putrescine in the rat liver within a few hours of treatment with carbon tetrachloride (1, 2) provides evidence supporting this hypothesis. This putrescine cannot have come from the decarboxylation of ornithine because its accumulation was not blocked by application of the ornithine decarboxylase inhibitor, a-difluoromethylornithine (3,5). In the present paper we have studied the changes in polyamine levels and in the activities of spermidine Nl-acetyltrans-
i Supported in part by Grants GM-26290 from the National Institutes of Health, by a Research Fellowship (to Hannu P&o) from the Research Council for National Sciences of the Academy of Finland, and a travel grant from the League of Finnish-American Societies Scholarship Foundation (thanks to Scandinavia grant). 2 To whom correspondence should be addressed. 0003-9861/82/100730-08$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
730
CARBON
TETRACHLORIDE
AND
ferase and polyamine oxidase brought about by carbon tetrachloride in the mouse liver. Much greater accumulation of the acetylated derivatives was observed in this species even though the induction of the acetylase occurred to exactly the same extent as in the rat. The greater accumulation appeared to be due to a much lower polyamine oxidase activity in the mouse liver. Very recently, Seiler and colleagues have also observed the accumulation of N’-acetylspermine in mouse liver after treatment with carbon tetrachloride (8). These observations provide strong support for the hypothesis that the acetylase and oxidase provide a system for interconversion of the polyamines that is usually limited by the activity of the acetylase. MATERIALS
AND
METHODS
[ucetyl-1-“C]Acetyl-CoA (49.8 mCi/mmol) and [tetramethylene-1,4-‘“Clspermidine (98.7 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts. Authentic acetylated polyamine standards were generous gifts from Dr. M. M. Abdel Monem, University of Minnesota, Minneapolis. All other biochemicals were obtained from Sigma Chemical Company, St. Louis, Missouri. Male mice of the Crl:CD-1 strain weighing about 30 g and male Sprague-Dawley rats weighing about 300 g were used in all experiments. The animals were allowed free access to water and a standard laboratory chow diet and were treated with carbon tetrachloride at a dose of 1 ml/kg at approximately 9:00 AM. The carbon tetrachloride was dissolved in sesame oil at a concentration such that the mice received an injection of about 0.3-0.5 ml and the rats received 0.5-1.0 ml. All injections were given intraperitoneally. Treatment with sesame oil alone did not alter polyamine metabolism. Tissue samples for enzyme assays were homogenized after removal from the animal. Extracts for assay of spermidine N’-acetyltransferase were prepared and assayed as previously described (2, 3). The assay medium contained 3 mM spermidine, 100 mM Tris-HCl, pH 7.8, and 8 PM [1-‘%]acetyl-CoA and was incubated for 10 min at 30°C. All assays were made under conditions in which the activity was proportional to the amount of protein added and to the time of incubation. Polyamine oxidase was assayed by the method described by Hiiltta (9) in which the putrescine product was separated from the labeled spermidine substrate by paper electrophoresis. The assays contained 100 mM glycine-NaOH buffer, pH 9.5,0.4 mbf[tetrumethylene-
RODENT
POLYAMINE
METABOLISM
731
1,4-‘“Clspermidine (1 pCi/rmol), 5 mM dithiothreitol, 5 mM benzaldehyde, and protein from a liver homogenate. The assay was carried out by incubation at 37°C for 30 min. Results were expressed as nanomoles of product produced per milligram of protein added and protein was determined by the methods of Bradford (10) using reagents from Bio-Rad Laboratories, Richmond, California, and bovine serum albumin as a standard. Polyamine analysis was carried out as previously described (2, 3) using an amino acid analyzer fitted with fluorescence detection and the elution conditions of (11). Tissue samples for polyamine measurement were frozen in liquid Nz immediately after removal and stored at -70°C until used. Frozen tissue was homogenized in 10 vol of 0.2 N perchloric acid and the protein was removed by centrifugation at 10,OOOg for 30 min. Aliquots of the supernatant were then used directly for the analysis. This procedure provided excellent separation of monoacetylputrescine, putrescine, monoacetylspermidines, spermidine, monoacetylspermine, and spermine from each other. However, monoacetylputrescine elutes very close to the position of certain amino acids and it may have been overestimated slightly. Also, although there was a partial resolution of the N’ and NE isomers of monoacetylspermidine, as previously observed by others using a similar ion-exchange column (12), these two substances overlapped. It appeared that the peak from tissue extracts of carbon tetrachloride-treated rodents corresponded to the N’-acetylspermidine isomer but it was necessary to confirm the absence of N8-acetylspermidine by other methods. This was accomplished by thin-layer chromatography of dansyl derivatives (13) or paper chromatography (14). It was also demonstrated that, when the samples were hydrolyzed in 6 N HCl, the materials identified as N’-acetylspermidine and N’-acetylspermine were converted as expected into spermidine and spermine. For the chromatographic separations and this hydrolysis, the liver samples were extracted with 5 vol of 5% trichloroacetic acid. After removal of the protein precipitate, the trichloroacetic acid was reversed by extraction with ether and the sample evaporated to dryness. Aliquots were subjected to acid hydrolysis (24 h in 6 N HCl in a sealed tube at 105°C) and then analyzed using the amino acid analyzer. Other aliquots were extracted with butanol(15) and subjected to paper electrophoresis in 0.1 M sodium citrate buffer, pH 3.6 or 4.3 (16,1’7), but this method revealed only three ninhydrin-positive spots corresponding to putrescine, spermidine, and spermine because N’acetylspermidine migrated at exactly the same rate as spermidine itself in this system. [This method which has been very widely used for the determination of polyamine concentrations (12,13) therefore overestimates spermidine content in any sample which contains N’-acetylspermidine.] The samples
732
P&&G AND PEGG
were, therefore, separated by descending paper chromatography using Whatman 3MM paper and l-propanol:triethylamine:water (353%) as solvent (14). The R, values were as follows: N*-acetylspermidine (&0.73), spermine (0.5’7), N’-acetylspermidine (0.32), spermidine (0.277, and putrescine (0.20). In order to completely resolve putrescine, spermidine, and N1acetylspermidine it was necessary to run the papers for 22 h which led to the elution of N*-acetylspermidine and spermine from the end of the paper. The latter substances were, therefore, measured in separate runs of 15 h. After electrophoresis or paper chromatography the polyamines were determined by staining with ninhydrin (15). Other aliquots were dansylated and the dansyl derivatives separated by thin-layer chromatography (13). Although this method is much more sensitive only a limited amount of extract could be processed and applied to the plate whereas a sample equivalent to a larger amount of liver could be separated by paper chromatography. The sensitivity of estimation was such that 5 nmol/ g liver of N*-acetylspermidine could have been detected, but none of the samples contained this amount of the N*-acetylspermidine isomer. The amount of N’-acetylspermidine determined by these methods was similar in all samples tested to that found using the amino acid analyzer.
HOURS
AFTER -Ccl,
FIG. 1. Activities of polyamine oxidase and spermidine iV’-acetyltransferase in rat and mouse liver after a single dose of carbon tetrachloride. Results are shown for rat polyamine oxidase (w), mouse polyamine oxidase (A), rat spermidine Ni-acetyltransferase (O), and mouse spermidine Nl-acetyltransferase (m). Results are shown +SD for at least four estimations at each point.
RESULTS
The effects of a single dose of carbon tetrachloride (1 ml/kg) on spermidine N’acetyltransferase and polyamine oxidase activities in extracts from rat and mouse liver are shown in Fig. 1. Spermidine N’acetyltransferase increased very rapidly in the mouse reaching a peak at 6 h after treatment at least 25 times greater than the control. The activity then fell rapidly but did not return totally to control values until 48 h. These changes are very similar to those seen previously in the rat liver after slightly larger doses of the hepatotoxin (2, 3) and, as shown in Fig. 1, in the present experiments, the same dose of carbon tetrachloride produced an almost equal increase in the activity in the rat. The only significant difference was that in the rat the activity declined somewhat more rapidly, reaching control values within 24 h. There was virtually no difference in either the basal or the peak values for the acetylase in the two species. As previously reported for the rat acetylase induced by carbon tetrachloride (3, 5), the mouse en-
zyme formed exclusively Nl-acetylspermidine when acting on spermidine as substrate. Putrescine was not a substrate for the induced acetylase (results not shown). In contrast to the rapid induction of acete, polyamine oxidase activity was not altered significantly in either species by treatment with carbon tetrachloride but the basal enzyme level in the mouse was only 5% of the activity in the rat (Fig. 1). Carbon tetrachloride produced large changes in the hepatic polyamine content in the mouse, which are detailed in Table I. N1-Acetylspermidine was below the limit of detection (about 5 nmol/g) in the control mice but rose rapidly to a peak of more than 600 nmol/g at 6 h, corresponding to the peak of acetylase activity. The identity of this material was confirmed as N’-acetylspermidine by (1) its comigration with authentic N1-acetylspermidine on paper electrophoresis or paper chromatography and on thin-layer chromatography (as a dansyl derivative) and by its hydrolysis to spermidine when heated in 6 N HCI (Table II). N*-Acetylspermidine
CARBON TETRACHLORIDE
AND RODENT POLYAMINE TABLE
EFFECT
733
METABOLISM
I
OF TREATMENT WITH CARBON TETRACHLORIDE ON CONTENT OF FREE ACET~LATED POLYAMINES IN MOUSE LIVER
AND
Content of polyamines (nmol/g wet wt) Hours after ccl,
Putrescine 12f 2 29 + 20 44f 8 91 + 41 123+40 177 +- 27 256 2 40 98 k 39
0
2 4 6 8 16 24 48
N’-AcetyIspermidine
N’-Acetylspermine
Spermidine
ND ND 262 f 35 611 f 238 285 f 64 135 f 45 145 f 48 ND
966 1021 943 609 536 459 533 1085
+ k + f + f f +
146 143 79 131 96 67 84 183
ND ND 15+ 5 45 f 15 152 5 ND ND ND
Spermine 1177 1066 1201 1140 889 699 435 354
f 75 +- 114 f 216 f 201 rf: 88 + 133 + 113 + 105
Note. The mice received carbon tetrachloride (1 ml/kg) at the time before death shown. Results are given as mean -t SD for at least five estimations. Ns-Acetylspermidine was not detected (~5 nmol/g) at any time point. Monoacetylputrescine was less than 10% of the putrescine content at all time points. ND, not detected (<5 nmol/g).
was not detected in any of the liver samples and the limit of detection was 5 nmol/ g. Monoacetylputrescine was found in some liver samples in amounts of 5-10s of the putrescine content, but because this substance may have been overestimated due to the proximity of much larger peaks corresponding to amino acids, this value is not included in Table II. However, since the content of monoactylputrescine was always less than 10% of the putrescine content, its contribution to the total polyamine content of the liver is negligible. TABLE EFFECT
OF ACID HYDROLYSIS
ON POLYAMINE
Also present in the samples obtained 6 h after treatment with carbon tetrachloride were small amounts (maximum 45 nmol/g) of material that was identified as N1-acetylspermine from its elution position and the fact that acid hydrolysis converted it to spermine. The treatment with carbon tetrachloride produced a substantial increase in hepatic putrescine in the mouse reaching peak values at 24 h which were 20 times the control value. Both spermidine and spermine declined by about 50% over the first 24 h but spermidine valII CONTENT
OF MOUSE
LIVER
EXTRACTS
Polyamine content (nmol/sample) Treatment Unhydrolyzed Hydrolyzed Unhydrolyzed Hydrolyzed
Source of sample Control Control CCL-treated CCL-treated
Putrescine 14 10 42 56
N’-Acetylspermidine
Spermidine 966 868 730 1109
Spermine 1200 1116 1160 997
Note. Tissue extracts were prepared from control rats and rats treated with carbon tetrachloride (0.1 ml/ kg) 6 h before death. The deproteinized extracts were analyzed directly or after hydrolysis for 24 h in 6 N HCl as described under Materials and Methods.
734
P&So
AND
PEGG
TABLE EFFECT
III
OF TREATMENT WITH CARBON TETRACHLORIDE ON CONTENT ACET~LATED POLYAMINES IN RAT LIVER Content
Hours after CCL 0 6 12 16 24 48
Putrescine 12+ 1 416 + 34 594 + 103 749 + 151 141 + 45 68+ 23
Note. Rats were treated with + SD for at least five estimations. ‘ND, not detected (~5 nmol/g). g) at any time point. Monoacetyl time points.
carbon
of polyamines
N’-Acetyispermidine
NE-Acetylspermidine putrescine was present
ues then recovered while spermine remained low. There was a significant net loss of polyamines from the liver such that the total of all the polyamines given in Table I at 24 h was only 64% of that in the controls. It should also be noted that there was a slight increase in the wet weight of the liver following carbon tetrachloride treatment presumably due to an increase in fluid intake. The mean liver weight increased from 1.92 to 2.21 g at 24 h but this increase is not enough to account for the fall of 36% in total polyamines. These changes in the mouse liver polyamines are in several ways different to those seen in the rat liver after treatment with the same dose of the hepatotoxin (Table III). In the rat no peak corresponding to W-acetylspermine was seen at any time. The maximal level of W-acetylspermidine that could be detected in the rat also occurred at 6 h, the peak time for acetylase activity, but the value was only 66 nmol/g, about 100th of that in the mouse. However, there was a much larger and more rapid increase in putrescine, which reached values 35 times greater than control at 6 h and peaked at 16 h at a value 62 times above control. [In the rat, as in the mouse, less than 10% of the putrescine was present as monoacetylpu-
(nmol/g
wet wt.)
Spermidine
ND” 66 + 33 20+ 9 ND ND ND tetrachloride
OF FREE AND
as described
803 444 421 724 876 1102
Spermine
f 100 + 136 + 141 + 179 + 161 z!z 148
in Table
I. Results
1310 1250 804 850 653 680 are
+ 160 + 83 f 175 + 38 + 93 Ik 114
given
as mean
and N’-acetylspermine were not detected (~5 nmol/ at less than 10% of the content of putrescine at all
trescine at all times.] The decline in spermidine was more transient in the rat and by 24 h spermidine had returned to control values although the spermine content decreased and remained at about 50% of the control value. There was a small reduction in total polyamine content in the rat but this amounted to only 20% at 24 h and could partly by accounted for by the small increase in liver wet weight. DISCUSSION
The measurements of polyamine oxidase activity described in Fig. 1 were carried out using spermidine in the presence of benzaldehyde as a substrate (9). Seiler and colleagues (6,18) have shown that this enzyme acts on N’-acetylspermidine and N1-acetylspermine at a rate about 12 times greater than with this substrate and that the Km values for these acetylated polyamines are 14 and 0.6 PM, respectively. Therefore, the polyamine oxidase activity in the rat liver appear to be capable of degrading 30 pmol acetylated polyamine/ h/g tissue and that in the mouse could degrade 1.5 pmol/h/g tissue. However, these values are likely to be substantial overestimates since (a) polyamine oxidase is partly located in peroxisomes (9) and
CARBON
TETRACHLORIDE
AND
access of the enzyme to the acetylated polyamine substrate may be limited; (b) the enzyme has a very high optimal pH; and (c) degradation of the acetylated polyamines is inhibited by free polyamines (6). The mouse liver after treatment with carbon tetrachloride contains the highest levels of acetylated polyamine derivatives so far reported in any mammalian tissue. Our results are in fairly good agreement in this respect with those reported for a single time point by Seiler et al. (8) although their value (174 nmol/g) for N’acetylspermidine at 6 h was considerably lower than ours. They also provided more rigorous evidence for the identification of N1-acetylspermine in the mouse livers after treatment with this hepatotoxin. It is known that the inducible spermidine N’acetyltransferase will also act on spermine as substrate in vitro (3) and, therefore, the detection of N’-acetylspermine in the liver at times when the acetylase activity is very high is not unexpected. The mouse liver system, therefore, provides an excellent model for studies of the function and disposition of the Nl-acetylated polyamine derivatives. At present, only one metabolic route for Nl-acetylspermidine is clearly established, namely, its conversion to putrescine by the action of polyamine oxidase (6,7). In the rat liver under all circumstances the activity of polyamine oxidase is so much greater than that of the acetylase that no detectable accumulation of N’-acetylspermine [which has a very low Km of 0.6 pM as a substrate for the oxidase (S)] and very little accumulation of N’-acetylspermidine can occur. At the time corresponding to the peak of acetylase activity the most striking increase is in the content of putrescine (Table II). In the mouse liver the activity of polyamine oxidase is much lower and this permits N’-acetylspermine and N’-acetylspermidine to accumulate to higher levels and delays the conversion of the latter into putrescine. Another possible fate for acetylated polyamines would be direct deacetylation and there is convincing evidence for the existence of an enzyme deacetylating NE-
RODENT
POLYAMINE
METABOLISM
735
acetylspermidine in rat liver (19, 20). The existence of this enzyme may be relevant to the absence of N8-acetylspermidine from the liver samples at all times, but since the inducible acetylase forms exclusively the N’-acetylspermidine isomer there is no reason to expect the appearance of N8-acetylspermidine. It has been claimed that rat liver contains a deacetylase active on N’-acetylspermidine (21), but we (3,22) and others (19, 23,25) have been unable to confirm this suggestion. In our experiments when crude rat liver extracts (3) or similar mouse liver extracts (unpublished observations) were incubated with N’-acetylspermidine the only detectable product of its degradation was putrescine confirming the original report of Blankenship (19,23). In the experiments in which deacetylation of Nl-acetylspermidine was claimed to occur, the products of the reaction were not characterized and the reaction was assayed by the conversion of N’-acetylspermidine labeled in the acetate moiety into a form extractable into ethyl acetate (21). This method does not establish that direct deacetylation takes place and it appears that oxidation by polyamine oxidase is quantitatively the major fate of N’-acetylspermidine (6, 18, 22, 23).
However, another metabolic route or transport of the acetylated derivative out of the cell may occur. Such transport or leakage from the cell would be in agreement with the finding that the total polyamine content of the liver decreases by almost 40% in the mouse liver after treatment with carbon tetrachloride, but this could also result from increased putrestine catabolism. Our finding that polyamine oxidase activity did not change significantly after treatment with carbon tetrachloride is in agreement with previous studies in the rat which showed either no change or only a small increase in the activity of liver polyamine oxidase after treatment with hepatotoxins, growth hormone, or partial hepatectomy (9, 24, 25). In contrast to spermidine Nl-acetyltransferase, which is highly inducible in the rat (2, 3, 5, 22, 26-28) and in the mouse (Fig.
736
P&L)
AND
l), and has a very short half-life (28), polyamine oxidase appears to be a relatively stable activity and the enzyme has a long half-life (24). The only net route for new putrescine production in mammalian cells is via the activity of ornithine decarboxylase and this enzyme also increases after treatment with carbon tetrachloride but more slowly than the spermidine N’-acetyltransferase (l-3). Up to 9 h after giving the hepatotoxin, virtually all of the putrescine is generated via the acetylase/oxidase system for degradation of the higher polyamines but at later times ornithine decarboxylase takes over this role and the polyamines can be resynthesized (3). The prolonged elevation of spermidine Nl-acetyltransferase in the mouse liver over the period of 16 to 24 h results in the continued production of N’-acetylspermidine from spermidine which is replenished by de nova synthesis. Therefore, the overall loss of polyamine from the liver may well be even more than the 40% based on the liver content reported in Table I. Finally, it remains to be determined why there is such a profound loss of spermine in the rodent livers after exposure to carbon tetrachloride. The major part of this reduction occurs after the peak of acetylase activity has declined in both rats and mice. It is, therefore, unlikely that the conversion of spermine to monoacetylspermine accounts for this fall even though monoacetylspermine is an excellent substrate for polyamine oxidase and has a very low Km (6, 7, 18), so that the levels of N’-acetylspermine actually found probably greatly underestimate its production even in the mouse. It is possible that part of the decline in spermine is due to a reduction in the normal rate of synthesis. In the presence of greatly elevated concentrations of putrescine, most of the available decarboxylated adenosylmethionine would be used by spermidine synthase to convert putrescine into spermidine (22,30,31). However, the reduction in synthesis is unlikely to produce such a large decrease in spermine within a few hours and other routes of degradation or
PEGG
transport out of the cell may also take place. The possibility that treatment with hepatotoxins renders the cell membrane leaky with respect to polyamines appears to be worth investigation. REFERENCES 1. H~L’ITA,
E., SINERVIRTA,
R., AND J&E,
J. (1973)
Biochem. Biophgs. Rea Gwnmun
54,350~357. 2. MATSUI, I., AND PEGG, A. E. (1980) Biochem Biophys Res. Commu~ 92,1009-1015. 3. MATSUI,
I., WIEGAND,
J. Bid
L., AND PEGG,
A. E. (1981)
Chem 256,2454-2459.
4. ABDEL-MONEM,
M. M., AND MERDINK,
J. L. (1981)
Life Sci 28,2017-2023. 5. PEGG, A. E., MATSUI, I., SEELY, J. E., PRITCHARD, M. L., AND P&o, H. (1981) Med Biol 59,327333. 6. BOLKENIUS, F. N., AND SEILER, N. (1981) Int. J. Biochem 13.287-292. 7. SUZUKI, O., MATSUMOTO, T., OYA, M., AND KATSUMATA, Y. (1981) Biochim Biophys. Acta 677, 190-193. 8. SEILER, N., BOLKENIUS, F. N., KNODGEN, B., AND HAEGELE, K. (1981) B&him Biophys. Actu 676, l-7. 9. H~LTT~, E. (1977) Biochemistry 16,91-100. 10. BRADFORD, M. M. (1976) Anal Biochem 72,248254. 11. SEIDENFELD, J., AND MARTON, L. J. (1979) Bb them Biophys Res. Commun 86,1192-1198. 12. FOLK, J. E., PARK, M. H., CHUNG, S. I., SCHRODE, J., LESTER, E. P., AND COOPER, H. L. (1980) J.
Biol Chem 255,3695-3700. 13. ABDEL-MONEM,
M. M.,
AND OHNO,
K. (1977)
J.
Phar7n sci 66.1195-1197. 14. DUBIN,
D. T., ANDROSENTHAL,
S. M. (1960)
J. Bid
Chem. 235, 776-782. 15. RAINA, A., (1963) Acta Chem Scand GO(Supp1. 218), 3-81. 16. RAINA, A., AND COHEN,
S. S. (1966)
Proc. Nat.
Acad Sci USA 55.1587-1593. 17. Posti, H., AND J&E, J. (1976) Biocherx J. 158, 485-488. 18. SEILER, N., BOLKENIUS, F. N., AND RENNERT, 0. M. (1981) Med Biol 59,334-346. 19. BLANKENSHIP, J. (1978) Arch. Biochem. Biqphys. 189.20-27. 20. SEILER, N. (1981) in Polyamines in Normal and Neoplastic Growth (Morris, D. R., and Marton, L. J., eds.), pp. 127-149, Dekker, New York. 21. LIBBY, P. R. (1978) Arch. Biochem Biuphys. 188, 360-363. H., MATSUI, I., AND BE22. PEGG, A. E., HIBASAMI, THELL, D. R. (1981) Advan Enzyme Regal. 19, 427-451.
CARBON TETRACHLORIDE 23. BLANKENSHIP. J. (1979) sot 22.115-118: 24. SEILER, N., B~LKENIUS, MAMONT, P. (1980)
AND RODENT POLYAMINE
Proc West. Phak F. N., KNODGEN,
Biochim Biophya
B., AND Acta 615.
25. SEILER, N., BOLKENIUS, F. N., AND KNODGEN, (1980) B&him Biophys. Actu 633,181-190. 26. MATSUI,
I., AND PEGG, A. E. (1980)
phys. Ada 633,87-94.
B&him
B.
Bb
737
27. DELLA RAGIONE, F., MATSUI, I., AND PEGG, (1982) Fed Prw 41.904. 28. MA&JI; I., AND PEGG, A. E. (1981) Biochem
A. E. Bti
phys. Acta 675,373-378. 29. J&E,
480-488.
METABOLISM
E., Pti,
H., ANDRAINA,
A. (19’78)
B&him
Biophzlg Acta 478,241~293. 30. WILLIAMS-ASHMAN,
H. G., ANDPEGG,
A. E. (1981)
in Polyamines in Normal and Neoplastic Growth (Morris, D. R., and Marton, L. J., eds.), pp. 3-42, Dekker, New York.
217, No. 2, September,
pp. 730-737,
1982
Effect of Carbon Tetrachloride on Polyamine Metabolism in Rodent Liver’ HANNU
P&is
ANTHONY
AND
E. PEGG’
Departmmzt of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, Pennsylvania State University, 500 University Drive, Hershey, Pennsylvania 17088 Received
April
30, 1982
Administration of hepatotoxic doses of carbon tetrachloride to mice produced a 25fold increase in spermidine/spermine Nr-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 loo-fold from levels below the limit of detection to 0.6 pmol of N1-acetylspermidine and 0.045 pmol 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 N’-acetyltransferase in this species, the rise in acetylated polyamines was much smaller and more transient. The content of N’-acetylspermidine was increased only to 0.066 pmol/g and N’-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 N’-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.
Polyamine metabolism in rat liver is dramatically affected by exposure to the hepatotoxin, carbon tetrachloride (l-3). Such exposure leads to the induction of an acetylase which acetylates spermidine and spermine at the N’ position (2, 3). Although this enzyme increases manyfold within a few hours, only small amounts of N’-acetylspermidine were detected in the liver at times corresponding to the peak of acetylase activity and monoace-
tylspermine was not detected at all (3-5). It was suggested (3,5) that the absence of large quantities of these acetylated derivates was due to their rapid oxidation by the enzyme polyamine oxidase which converts monoacetylspermine to spermidine and N’-acetylspermidine into putrescine (6, 7). The striking accumulation of putrescine in the rat liver within a few hours of treatment with carbon tetrachloride (1, 2) provides evidence supporting this hypothesis. This putrescine cannot have come from the decarboxylation of ornithine because its accumulation was not blocked by application of the ornithine decarboxylase inhibitor, a-difluoromethylornithine (3,5). In the present paper we have studied the changes in polyamine levels and in the activities of spermidine Nl-acetyltrans-
i Supported in part by Grants GM-26290 from the National Institutes of Health, by a Research Fellowship (to Hannu P&o) from the Research Council for National Sciences of the Academy of Finland, and a travel grant from the League of Finnish-American Societies Scholarship Foundation (thanks to Scandinavia grant). 2 To whom correspondence should be addressed. 0003-9861/82/100730-08$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
730
CARBON
TETRACHLORIDE
AND
ferase and polyamine oxidase brought about by carbon tetrachloride in the mouse liver. Much greater accumulation of the acetylated derivatives was observed in this species even though the induction of the acetylase occurred to exactly the same extent as in the rat. The greater accumulation appeared to be due to a much lower polyamine oxidase activity in the mouse liver. Very recently, Seiler and colleagues have also observed the accumulation of N’-acetylspermine in mouse liver after treatment with carbon tetrachloride (8). These observations provide strong support for the hypothesis that the acetylase and oxidase provide a system for interconversion of the polyamines that is usually limited by the activity of the acetylase. MATERIALS
AND
METHODS
[ucetyl-1-“C]Acetyl-CoA (49.8 mCi/mmol) and [tetramethylene-1,4-‘“Clspermidine (98.7 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts. Authentic acetylated polyamine standards were generous gifts from Dr. M. M. Abdel Monem, University of Minnesota, Minneapolis. All other biochemicals were obtained from Sigma Chemical Company, St. Louis, Missouri. Male mice of the Crl:CD-1 strain weighing about 30 g and male Sprague-Dawley rats weighing about 300 g were used in all experiments. The animals were allowed free access to water and a standard laboratory chow diet and were treated with carbon tetrachloride at a dose of 1 ml/kg at approximately 9:00 AM. The carbon tetrachloride was dissolved in sesame oil at a concentration such that the mice received an injection of about 0.3-0.5 ml and the rats received 0.5-1.0 ml. All injections were given intraperitoneally. Treatment with sesame oil alone did not alter polyamine metabolism. Tissue samples for enzyme assays were homogenized after removal from the animal. Extracts for assay of spermidine N’-acetyltransferase were prepared and assayed as previously described (2, 3). The assay medium contained 3 mM spermidine, 100 mM Tris-HCl, pH 7.8, and 8 PM [1-‘%]acetyl-CoA and was incubated for 10 min at 30°C. All assays were made under conditions in which the activity was proportional to the amount of protein added and to the time of incubation. Polyamine oxidase was assayed by the method described by Hiiltta (9) in which the putrescine product was separated from the labeled spermidine substrate by paper electrophoresis. The assays contained 100 mM glycine-NaOH buffer, pH 9.5,0.4 mbf[tetrumethylene-
RODENT
POLYAMINE
METABOLISM
731
1,4-‘“Clspermidine (1 pCi/rmol), 5 mM dithiothreitol, 5 mM benzaldehyde, and protein from a liver homogenate. The assay was carried out by incubation at 37°C for 30 min. Results were expressed as nanomoles of product produced per milligram of protein added and protein was determined by the methods of Bradford (10) using reagents from Bio-Rad Laboratories, Richmond, California, and bovine serum albumin as a standard. Polyamine analysis was carried out as previously described (2, 3) using an amino acid analyzer fitted with fluorescence detection and the elution conditions of (11). Tissue samples for polyamine measurement were frozen in liquid Nz immediately after removal and stored at -70°C until used. Frozen tissue was homogenized in 10 vol of 0.2 N perchloric acid and the protein was removed by centrifugation at 10,OOOg for 30 min. Aliquots of the supernatant were then used directly for the analysis. This procedure provided excellent separation of monoacetylputrescine, putrescine, monoacetylspermidines, spermidine, monoacetylspermine, and spermine from each other. However, monoacetylputrescine elutes very close to the position of certain amino acids and it may have been overestimated slightly. Also, although there was a partial resolution of the N’ and NE isomers of monoacetylspermidine, as previously observed by others using a similar ion-exchange column (12), these two substances overlapped. It appeared that the peak from tissue extracts of carbon tetrachloride-treated rodents corresponded to the N’-acetylspermidine isomer but it was necessary to confirm the absence of N8-acetylspermidine by other methods. This was accomplished by thin-layer chromatography of dansyl derivatives (13) or paper chromatography (14). It was also demonstrated that, when the samples were hydrolyzed in 6 N HCl, the materials identified as N’-acetylspermidine and N’-acetylspermine were converted as expected into spermidine and spermine. For the chromatographic separations and this hydrolysis, the liver samples were extracted with 5 vol of 5% trichloroacetic acid. After removal of the protein precipitate, the trichloroacetic acid was reversed by extraction with ether and the sample evaporated to dryness. Aliquots were subjected to acid hydrolysis (24 h in 6 N HCl in a sealed tube at 105°C) and then analyzed using the amino acid analyzer. Other aliquots were extracted with butanol(15) and subjected to paper electrophoresis in 0.1 M sodium citrate buffer, pH 3.6 or 4.3 (16,1’7), but this method revealed only three ninhydrin-positive spots corresponding to putrescine, spermidine, and spermine because N’acetylspermidine migrated at exactly the same rate as spermidine itself in this system. [This method which has been very widely used for the determination of polyamine concentrations (12,13) therefore overestimates spermidine content in any sample which contains N’-acetylspermidine.] The samples
732
P&&G AND PEGG
were, therefore, separated by descending paper chromatography using Whatman 3MM paper and l-propanol:triethylamine:water (353%) as solvent (14). The R, values were as follows: N*-acetylspermidine (&0.73), spermine (0.5’7), N’-acetylspermidine (0.32), spermidine (0.277, and putrescine (0.20). In order to completely resolve putrescine, spermidine, and N1acetylspermidine it was necessary to run the papers for 22 h which led to the elution of N*-acetylspermidine and spermine from the end of the paper. The latter substances were, therefore, measured in separate runs of 15 h. After electrophoresis or paper chromatography the polyamines were determined by staining with ninhydrin (15). Other aliquots were dansylated and the dansyl derivatives separated by thin-layer chromatography (13). Although this method is much more sensitive only a limited amount of extract could be processed and applied to the plate whereas a sample equivalent to a larger amount of liver could be separated by paper chromatography. The sensitivity of estimation was such that 5 nmol/ g liver of N*-acetylspermidine could have been detected, but none of the samples contained this amount of the N*-acetylspermidine isomer. The amount of N’-acetylspermidine determined by these methods was similar in all samples tested to that found using the amino acid analyzer.
HOURS
AFTER -Ccl,
FIG. 1. Activities of polyamine oxidase and spermidine iV’-acetyltransferase in rat and mouse liver after a single dose of carbon tetrachloride. Results are shown for rat polyamine oxidase (w), mouse polyamine oxidase (A), rat spermidine Ni-acetyltransferase (O), and mouse spermidine Nl-acetyltransferase (m). Results are shown +SD for at least four estimations at each point.
RESULTS
The effects of a single dose of carbon tetrachloride (1 ml/kg) on spermidine N’acetyltransferase and polyamine oxidase activities in extracts from rat and mouse liver are shown in Fig. 1. Spermidine N’acetyltransferase increased very rapidly in the mouse reaching a peak at 6 h after treatment at least 25 times greater than the control. The activity then fell rapidly but did not return totally to control values until 48 h. These changes are very similar to those seen previously in the rat liver after slightly larger doses of the hepatotoxin (2, 3) and, as shown in Fig. 1, in the present experiments, the same dose of carbon tetrachloride produced an almost equal increase in the activity in the rat. The only significant difference was that in the rat the activity declined somewhat more rapidly, reaching control values within 24 h. There was virtually no difference in either the basal or the peak values for the acetylase in the two species. As previously reported for the rat acetylase induced by carbon tetrachloride (3, 5), the mouse en-
zyme formed exclusively Nl-acetylspermidine when acting on spermidine as substrate. Putrescine was not a substrate for the induced acetylase (results not shown). In contrast to the rapid induction of acete, polyamine oxidase activity was not altered significantly in either species by treatment with carbon tetrachloride but the basal enzyme level in the mouse was only 5% of the activity in the rat (Fig. 1). Carbon tetrachloride produced large changes in the hepatic polyamine content in the mouse, which are detailed in Table I. N1-Acetylspermidine was below the limit of detection (about 5 nmol/g) in the control mice but rose rapidly to a peak of more than 600 nmol/g at 6 h, corresponding to the peak of acetylase activity. The identity of this material was confirmed as N’-acetylspermidine by (1) its comigration with authentic N1-acetylspermidine on paper electrophoresis or paper chromatography and on thin-layer chromatography (as a dansyl derivative) and by its hydrolysis to spermidine when heated in 6 N HCI (Table II). N*-Acetylspermidine
CARBON TETRACHLORIDE
AND RODENT POLYAMINE TABLE
EFFECT
733
METABOLISM
I
OF TREATMENT WITH CARBON TETRACHLORIDE ON CONTENT OF FREE ACET~LATED POLYAMINES IN MOUSE LIVER
AND
Content of polyamines (nmol/g wet wt) Hours after ccl,
Putrescine 12f 2 29 + 20 44f 8 91 + 41 123+40 177 +- 27 256 2 40 98 k 39
0
2 4 6 8 16 24 48
N’-AcetyIspermidine
N’-Acetylspermine
Spermidine
ND ND 262 f 35 611 f 238 285 f 64 135 f 45 145 f 48 ND
966 1021 943 609 536 459 533 1085
+ k + f + f f +
146 143 79 131 96 67 84 183
ND ND 15+ 5 45 f 15 152 5 ND ND ND
Spermine 1177 1066 1201 1140 889 699 435 354
f 75 +- 114 f 216 f 201 rf: 88 + 133 + 113 + 105
Note. The mice received carbon tetrachloride (1 ml/kg) at the time before death shown. Results are given as mean -t SD for at least five estimations. Ns-Acetylspermidine was not detected (~5 nmol/g) at any time point. Monoacetylputrescine was less than 10% of the putrescine content at all time points. ND, not detected (<5 nmol/g).
was not detected in any of the liver samples and the limit of detection was 5 nmol/ g. Monoacetylputrescine was found in some liver samples in amounts of 5-10s of the putrescine content, but because this substance may have been overestimated due to the proximity of much larger peaks corresponding to amino acids, this value is not included in Table II. However, since the content of monoactylputrescine was always less than 10% of the putrescine content, its contribution to the total polyamine content of the liver is negligible. TABLE EFFECT
OF ACID HYDROLYSIS
ON POLYAMINE
Also present in the samples obtained 6 h after treatment with carbon tetrachloride were small amounts (maximum 45 nmol/g) of material that was identified as N1-acetylspermine from its elution position and the fact that acid hydrolysis converted it to spermine. The treatment with carbon tetrachloride produced a substantial increase in hepatic putrescine in the mouse reaching peak values at 24 h which were 20 times the control value. Both spermidine and spermine declined by about 50% over the first 24 h but spermidine valII CONTENT
OF MOUSE
LIVER
EXTRACTS
Polyamine content (nmol/sample) Treatment Unhydrolyzed Hydrolyzed Unhydrolyzed Hydrolyzed
Source of sample Control Control CCL-treated CCL-treated
Putrescine 14 10 42 56
N’-Acetylspermidine
Spermidine 966 868 730 1109
Spermine 1200 1116 1160 997
Note. Tissue extracts were prepared from control rats and rats treated with carbon tetrachloride (0.1 ml/ kg) 6 h before death. The deproteinized extracts were analyzed directly or after hydrolysis for 24 h in 6 N HCl as described under Materials and Methods.
734
P&So
AND
PEGG
TABLE EFFECT
III
OF TREATMENT WITH CARBON TETRACHLORIDE ON CONTENT ACET~LATED POLYAMINES IN RAT LIVER Content
Hours after CCL 0 6 12 16 24 48
Putrescine 12+ 1 416 + 34 594 + 103 749 + 151 141 + 45 68+ 23
Note. Rats were treated with + SD for at least five estimations. ‘ND, not detected (~5 nmol/g). g) at any time point. Monoacetyl time points.
carbon
of polyamines
N’-Acetyispermidine
NE-Acetylspermidine putrescine was present
ues then recovered while spermine remained low. There was a significant net loss of polyamines from the liver such that the total of all the polyamines given in Table I at 24 h was only 64% of that in the controls. It should also be noted that there was a slight increase in the wet weight of the liver following carbon tetrachloride treatment presumably due to an increase in fluid intake. The mean liver weight increased from 1.92 to 2.21 g at 24 h but this increase is not enough to account for the fall of 36% in total polyamines. These changes in the mouse liver polyamines are in several ways different to those seen in the rat liver after treatment with the same dose of the hepatotoxin (Table III). In the rat no peak corresponding to W-acetylspermine was seen at any time. The maximal level of W-acetylspermidine that could be detected in the rat also occurred at 6 h, the peak time for acetylase activity, but the value was only 66 nmol/g, about 100th of that in the mouse. However, there was a much larger and more rapid increase in putrescine, which reached values 35 times greater than control at 6 h and peaked at 16 h at a value 62 times above control. [In the rat, as in the mouse, less than 10% of the putrescine was present as monoacetylpu-
(nmol/g
wet wt.)
Spermidine
ND” 66 + 33 20+ 9 ND ND ND tetrachloride
OF FREE AND
as described
803 444 421 724 876 1102
Spermine
f 100 + 136 + 141 + 179 + 161 z!z 148
in Table
I. Results
1310 1250 804 850 653 680 are
+ 160 + 83 f 175 + 38 + 93 Ik 114
given
as mean
and N’-acetylspermine were not detected (~5 nmol/ at less than 10% of the content of putrescine at all
trescine at all times.] The decline in spermidine was more transient in the rat and by 24 h spermidine had returned to control values although the spermine content decreased and remained at about 50% of the control value. There was a small reduction in total polyamine content in the rat but this amounted to only 20% at 24 h and could partly by accounted for by the small increase in liver wet weight. DISCUSSION
The measurements of polyamine oxidase activity described in Fig. 1 were carried out using spermidine in the presence of benzaldehyde as a substrate (9). Seiler and colleagues (6,18) have shown that this enzyme acts on N’-acetylspermidine and N1-acetylspermine at a rate about 12 times greater than with this substrate and that the Km values for these acetylated polyamines are 14 and 0.6 PM, respectively. Therefore, the polyamine oxidase activity in the rat liver appear to be capable of degrading 30 pmol acetylated polyamine/ h/g tissue and that in the mouse could degrade 1.5 pmol/h/g tissue. However, these values are likely to be substantial overestimates since (a) polyamine oxidase is partly located in peroxisomes (9) and
CARBON
TETRACHLORIDE
AND
access of the enzyme to the acetylated polyamine substrate may be limited; (b) the enzyme has a very high optimal pH; and (c) degradation of the acetylated polyamines is inhibited by free polyamines (6). The mouse liver after treatment with carbon tetrachloride contains the highest levels of acetylated polyamine derivatives so far reported in any mammalian tissue. Our results are in fairly good agreement in this respect with those reported for a single time point by Seiler et al. (8) although their value (174 nmol/g) for N’acetylspermidine at 6 h was considerably lower than ours. They also provided more rigorous evidence for the identification of N1-acetylspermine in the mouse livers after treatment with this hepatotoxin. It is known that the inducible spermidine N’acetyltransferase will also act on spermine as substrate in vitro (3) and, therefore, the detection of N’-acetylspermine in the liver at times when the acetylase activity is very high is not unexpected. The mouse liver system, therefore, provides an excellent model for studies of the function and disposition of the Nl-acetylated polyamine derivatives. At present, only one metabolic route for Nl-acetylspermidine is clearly established, namely, its conversion to putrescine by the action of polyamine oxidase (6,7). In the rat liver under all circumstances the activity of polyamine oxidase is so much greater than that of the acetylase that no detectable accumulation of N’-acetylspermine [which has a very low Km of 0.6 pM as a substrate for the oxidase (S)] and very little accumulation of N’-acetylspermidine can occur. At the time corresponding to the peak of acetylase activity the most striking increase is in the content of putrescine (Table II). In the mouse liver the activity of polyamine oxidase is much lower and this permits N’-acetylspermine and N’-acetylspermidine to accumulate to higher levels and delays the conversion of the latter into putrescine. Another possible fate for acetylated polyamines would be direct deacetylation and there is convincing evidence for the existence of an enzyme deacetylating NE-
RODENT
POLYAMINE
METABOLISM
735
acetylspermidine in rat liver (19, 20). The existence of this enzyme may be relevant to the absence of N8-acetylspermidine from the liver samples at all times, but since the inducible acetylase forms exclusively the N’-acetylspermidine isomer there is no reason to expect the appearance of N8-acetylspermidine. It has been claimed that rat liver contains a deacetylase active on N’-acetylspermidine (21), but we (3,22) and others (19, 23,25) have been unable to confirm this suggestion. In our experiments when crude rat liver extracts (3) or similar mouse liver extracts (unpublished observations) were incubated with N’-acetylspermidine the only detectable product of its degradation was putrescine confirming the original report of Blankenship (19,23). In the experiments in which deacetylation of Nl-acetylspermidine was claimed to occur, the products of the reaction were not characterized and the reaction was assayed by the conversion of N’-acetylspermidine labeled in the acetate moiety into a form extractable into ethyl acetate (21). This method does not establish that direct deacetylation takes place and it appears that oxidation by polyamine oxidase is quantitatively the major fate of N’-acetylspermidine (6, 18, 22, 23).
However, another metabolic route or transport of the acetylated derivative out of the cell may occur. Such transport or leakage from the cell would be in agreement with the finding that the total polyamine content of the liver decreases by almost 40% in the mouse liver after treatment with carbon tetrachloride, but this could also result from increased putrestine catabolism. Our finding that polyamine oxidase activity did not change significantly after treatment with carbon tetrachloride is in agreement with previous studies in the rat which showed either no change or only a small increase in the activity of liver polyamine oxidase after treatment with hepatotoxins, growth hormone, or partial hepatectomy (9, 24, 25). In contrast to spermidine Nl-acetyltransferase, which is highly inducible in the rat (2, 3, 5, 22, 26-28) and in the mouse (Fig.
736
P&L)
AND
l), and has a very short half-life (28), polyamine oxidase appears to be a relatively stable activity and the enzyme has a long half-life (24). The only net route for new putrescine production in mammalian cells is via the activity of ornithine decarboxylase and this enzyme also increases after treatment with carbon tetrachloride but more slowly than the spermidine N’-acetyltransferase (l-3). Up to 9 h after giving the hepatotoxin, virtually all of the putrescine is generated via the acetylase/oxidase system for degradation of the higher polyamines but at later times ornithine decarboxylase takes over this role and the polyamines can be resynthesized (3). The prolonged elevation of spermidine Nl-acetyltransferase in the mouse liver over the period of 16 to 24 h results in the continued production of N’-acetylspermidine from spermidine which is replenished by de nova synthesis. Therefore, the overall loss of polyamine from the liver may well be even more than the 40% based on the liver content reported in Table I. Finally, it remains to be determined why there is such a profound loss of spermine in the rodent livers after exposure to carbon tetrachloride. The major part of this reduction occurs after the peak of acetylase activity has declined in both rats and mice. It is, therefore, unlikely that the conversion of spermine to monoacetylspermine accounts for this fall even though monoacetylspermine is an excellent substrate for polyamine oxidase and has a very low Km (6, 7, 18), so that the levels of N’-acetylspermine actually found probably greatly underestimate its production even in the mouse. It is possible that part of the decline in spermine is due to a reduction in the normal rate of synthesis. In the presence of greatly elevated concentrations of putrescine, most of the available decarboxylated adenosylmethionine would be used by spermidine synthase to convert putrescine into spermidine (22,30,31). However, the reduction in synthesis is unlikely to produce such a large decrease in spermine within a few hours and other routes of degradation or
PEGG
transport out of the cell may also take place. The possibility that treatment with hepatotoxins renders the cell membrane leaky with respect to polyamines appears to be worth investigation. REFERENCES 1. H~L’ITA,
E., SINERVIRTA,
R., AND J&E,
J. (1973)
Biochem. Biophgs. Rea Gwnmun
54,350~357. 2. MATSUI, I., AND PEGG, A. E. (1980) Biochem Biophys Res. Commu~ 92,1009-1015. 3. MATSUI,
I., WIEGAND,
J. Bid
L., AND PEGG,
A. E. (1981)
Chem 256,2454-2459.
4. ABDEL-MONEM,
M. M., AND MERDINK,
J. L. (1981)
Life Sci 28,2017-2023. 5. PEGG, A. E., MATSUI, I., SEELY, J. E., PRITCHARD, M. L., AND P&o, H. (1981) Med Biol 59,327333. 6. BOLKENIUS, F. N., AND SEILER, N. (1981) Int. J. Biochem 13.287-292. 7. SUZUKI, O., MATSUMOTO, T., OYA, M., AND KATSUMATA, Y. (1981) Biochim Biophys. Acta 677, 190-193. 8. SEILER, N., BOLKENIUS, F. N., KNODGEN, B., AND HAEGELE, K. (1981) B&him Biophys. Actu 676, l-7. 9. H~LTT~, E. (1977) Biochemistry 16,91-100. 10. BRADFORD, M. M. (1976) Anal Biochem 72,248254. 11. SEIDENFELD, J., AND MARTON, L. J. (1979) Bb them Biophys Res. Commun 86,1192-1198. 12. FOLK, J. E., PARK, M. H., CHUNG, S. I., SCHRODE, J., LESTER, E. P., AND COOPER, H. L. (1980) J.
Biol Chem 255,3695-3700. 13. ABDEL-MONEM,
M. M.,
AND OHNO,
K. (1977)
J.
Phar7n sci 66.1195-1197. 14. DUBIN,
D. T., ANDROSENTHAL,
S. M. (1960)
J. Bid
Chem. 235, 776-782. 15. RAINA, A., (1963) Acta Chem Scand GO(Supp1. 218), 3-81. 16. RAINA, A., AND COHEN,
S. S. (1966)
Proc. Nat.
Acad Sci USA 55.1587-1593. 17. Posti, H., AND J&E, J. (1976) Biocherx J. 158, 485-488. 18. SEILER, N., BOLKENIUS, F. N., AND RENNERT, 0. M. (1981) Med Biol 59,334-346. 19. BLANKENSHIP, J. (1978) Arch. Biochem. Biqphys. 189.20-27. 20. SEILER, N. (1981) in Polyamines in Normal and Neoplastic Growth (Morris, D. R., and Marton, L. J., eds.), pp. 127-149, Dekker, New York. 21. LIBBY, P. R. (1978) Arch. Biochem Biuphys. 188, 360-363. H., MATSUI, I., AND BE22. PEGG, A. E., HIBASAMI, THELL, D. R. (1981) Advan Enzyme Regal. 19, 427-451.
CARBON TETRACHLORIDE 23. BLANKENSHIP. J. (1979) sot 22.115-118: 24. SEILER, N., B~LKENIUS, MAMONT, P. (1980)
AND RODENT POLYAMINE
Proc West. Phak F. N., KNODGEN,
Biochim Biophya
B., AND Acta 615.
25. SEILER, N., BOLKENIUS, F. N., AND KNODGEN, (1980) B&him Biophys. Actu 633,181-190. 26. MATSUI,
I., AND PEGG, A. E. (1980)
phys. Ada 633,87-94.
B&him
B.
Bb
737
27. DELLA RAGIONE, F., MATSUI, I., AND PEGG, (1982) Fed Prw 41.904. 28. MA&JI; I., AND PEGG, A. E. (1981) Biochem
A. E. Bti
phys. Acta 675,373-378. 29. J&E,
480-488.
METABOLISM
E., Pti,
H., ANDRAINA,
A. (19’78)
B&him
Biophzlg Acta 478,241~293. 30. WILLIAMS-ASHMAN,
H. G., ANDPEGG,
A. E. (1981)
in Polyamines in Normal and Neoplastic Growth (Morris, D. R., and Marton, L. J., eds.), pp. 3-42, Dekker, New York.