chain-fluorinated polyamines as tumor markers

10
J Cancer Res Clin Oncol (1988) 114:71-80 Cancer Research and Clinical @ncology Springer-Verlag 1988 Chain-fluorinated polyamines as tumor markers * II. Metabolic aspects in normal tissues N. Seiler, S. Sarhan, B. Kn~idgen, and F. Gerhart Merrell Dow Research Institute, Strasbourg-Center, 16, rue d'Ankara, F-67084 Strasbourg C6dex, France Summary. The objective of this work was to study cer- tain metabolic aspects of fluorine-substituted analo- gues of natural polyamines in healthy experimental animals, with the aim of exploring their potential ap- plication as tumor markers. Tissue polyamine concen- trations were more effectively depleted by combined treatment with D,L-e-difluoromethylornithine, an irre- versible inhibitor of ornithine decarboxylase, and N1,N4-bis-allenylputrescine, an inactivator of poly- amine oxidase, than with either inhibitor alone. This suggests the general importance of polyamine inter- conversion as a metabolic source of putrescine. Ad- ministration of 2,2-difluoroputrescine after 2 weeks pretreatment with the two inhibitors caused the for- mation of 6,6-difluorospermidine and 6,6-difluoro- spermidine in nearly all tissues. Highest concentrations of the chain-fluorinated polyamines were observed in the small intestine. At 24 h after 2,2-difluoropu- trescine administration the amount was about 8% of the normal endogenous polyamine pool in the small intestine, but lower in all other tissues. Replenishment of endogenous polyamine pools is a relatively slow process. Approximately 9 days after cessation of treat- ment with the two inhibitors normal values had been reestablished. The rate of formation of endogenous polyamines was not affected by the presence of their difluoro analogues. Elimination of the chain-fluori- nated polyamines from tissues seems not to follow normal polyamine metabolic patterns. Their most rapid elimination coincides with the enhancement of endogenous polyamines, indicating that the fluoro analogues are displaced by the natural polyamines. Most of the 2,2-difluoroputrescine was rapidly ex- creted in the urine, and formation of a conjugate was detected. 6,6-Difluorospermidine was also a urinary * Dedicated to Professor Werner Kunz on the occasion of his 65th birthday Offprint requests to: N. Seiler excretion product. However, the metabolic fate of 6,6- difluorospermine could not be clarified. It was not found in urine, either free or as conjugate. The rela- tively low accumulation of chain-fluorinated poly- amines, together with their rapid elimination from normal tissues are characteristics which together with their previously established selective uptake into rap- idly proliferating tissues recommend them as potential tumor markers that can be determined by 19F-NMR spectroscopy. Key words" Chain-fluorinated polyamines - Tumor markers - Normal tissue Introduction 2,2-Difluoroputrescine (DFPut) is a substrate of sper- midine synthase (Gerhart et al. 1987). After its admin- istration to experimental animals 6,6-difluorosper- midine (6,6DFSpd) and 6,6-difluorospermine (6,6DFSpm) may be formed. The formation of chain- fluorinated polyamines was considerably enhanced if tissue concentrations of putrescine and spermidine were decreased by pretreatment with the ornithine de- carboxylase (ODC) inhibitor D,L-e-difluoromethylor- nithine (DFMO). Since polyamine depletion with DFMO was especially effective in rapidly proliferat- ing tissues (such as experimental solid tumors, small intestines, and chick embryos) with their usually high ODC activity, a rather selective accumulation of fluorinated polyamines could be achieved in these tis- sues (Sarhan et al. 1987), in analogy to the enhanced accumulation of cadaverine and formation of N-(3- aminopropyl)cadaverine and N,N'-bis-(3-aminopro- pyl)cadaverine by DFMO-treated tumor cells (Mamont et al. 1978; Alhonen-Hongisto et al. 1982).

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J Cancer Res Clin Oncol (1988) 114:71-80 Cancer Research and

Clinical @ncology �9 Springer-Verlag 1988

Chain-fluorinated polyamines as tumor markers *

II. Metabolic aspects in normal tissues

N. Seiler, S. Sarhan, B. Kn~idgen, and F. Gerhart Merrell Dow Research Institute, Strasbourg-Center, 16, rue d'Ankara, F-67084 Strasbourg C6dex, France

Summary. The objective of this work was to study cer- tain metabolic aspects of fluorine-substituted analo- gues of natural polyamines in healthy experimental animals, with the aim of exploring their potential ap- plication as tumor markers. Tissue polyamine concen- trations were more effectively depleted by combined treatment with D,L-e-difluoromethylornithine, an irre- versible inhibitor of ornithine decarboxylase, and N1,N4-bis-allenylputrescine, an inactivator of poly- amine oxidase, than with either inhibitor alone. This suggests the general importance of polyamine inter- conversion as a metabolic source of putrescine. Ad- ministration of 2,2-difluoroputrescine after 2 weeks pretreatment with the two inhibitors caused the for- mation of 6,6-difluorospermidine and 6,6-difluoro- spermidine in nearly all tissues. Highest concentrations of the chain-fluorinated polyamines were observed in the small intestine. At 24 h after 2,2-difluoropu- trescine administration the amount was about 8% of the normal endogenous polyamine pool in the small intestine, but lower in all other tissues. Replenishment of endogenous polyamine pools is a relatively slow process. Approximately 9 days after cessation of treat- ment with the two inhibitors normal values had been reestablished. The rate of formation of endogenous polyamines was not affected by the presence of their difluoro analogues. Elimination of the chain-fluori- nated polyamines from tissues seems not to follow normal polyamine metabolic patterns. Their most rapid elimination coincides with the enhancement of endogenous polyamines, indicating that the fluoro analogues are displaced by the natural polyamines. Most of the 2,2-difluoroputrescine was rapidly ex- creted in the urine, and formation of a conjugate was detected. 6,6-Difluorospermidine was also a urinary

* Dedicated to Professor Werner Kunz on the occasion of his 65th birthday

Offprint requests to: N. Seiler

excretion product. However, the metabolic fate of 6,6- difluorospermine could not be clarified. It was not found in urine, either free or as conjugate. The rela- tively low accumulation of chain-fluorinated poly- amines, together with their rapid elimination from normal tissues are characteristics which together with their previously established selective uptake into rap- idly proliferating tissues recommend them as potential tumor markers that can be determined by 19F-NMR spectroscopy.

Key words" Chain-fluorinated polyamines - Tumor markers - Normal tissue

Introduction

2,2-Difluoroputrescine (DFPut) is a substrate of sper- midine synthase (Gerhart et al. 1987). After its admin- istration to experimental animals 6,6-difluorosper- midine (6,6DFSpd) and 6,6-difluorospermine (6,6DFSpm) may be formed. The formation of chain- fluorinated polyamines was considerably enhanced if tissue concentrations of putrescine and spermidine were decreased by pretreatment with the ornithine de- carboxylase (ODC) inhibitor D,L-e-difluoromethylor- nithine (DFMO). Since polyamine depletion with DFMO was especially effective in rapidly proliferat- ing tissues (such as experimental solid tumors, small intestines, and chick embryos) with their usually high ODC activity, a rather selective accumulation of fluorinated polyamines could be achieved in these tis- sues (Sarhan et al. 1987), in analogy to the enhanced accumulation of cadaverine and formation of N-(3- aminopropyl)cadaverine and N,N'-bis-(3-aminopro- pyl)cadaverine by DFMO-treated tumor cells (Mamont et al. 1978; Alhonen-Hongisto et al. 1982).

72 N. Seller et al.: Chain-fluorinated polyamines as tumor markers. II.

I n contrast , nong rowing tissues produce only a certain p ropo r t i on of putrescine by decarboxyla t ion of orni thine. Significant amoun t s are formed by deg- r ada t ion of spermidine a long the so-called po lyamine in terconvers ion pa thway (Seller et al. 1985 a). In agree- men t with this fact spermidine concent ra t ions in liver, kidney, and b ra in could be lowered more effectively if bo th ODC, and po lyamine oxidase (PAO) were simul- taneously inhibi ted over an extended per iod of t ime (Bolkenius and Seller 1987).

Wi th the new possibili ty of deplet ing tissue sper- midine levels of nonpro l i fe ra t ing organs of mice by 50% or more, we a t tempted to ob ta in answers to the following questions:

(a) How rapidly are tissue po lyamine pools re- plenished after cessation of combined t rea tment with inhibi tors o f PAO and ODC?

(b) To what extent are the na tu ra l polyamines subst i tu ted in no rma l tissues by their dif luoro analo- gues, if D F P u t is adminis tered as a precursor?

(c) W h a t is the rate of e l iminat ion of f luor ina ted polyamines f rom no rma l tissues, and how is their e l imina t ion related to the res tora t ion of physiological po lyamine pat terns?

(d) W h a t are the excretory forms of the chain- f luor inated polyamines?

All these quest ions are per t inen t to the potent ia l use of cha in- f luor ina ted polyamines as t umor markers (Sarhan et al. 1987).

Materials

Usual laboratory chemicals (A grade) including aminoguanidine sul- phate were obtained from Baker Chemicals (Deventer, The Nether- lands) or Merck (Darmstadt, FRG). The DFMO (Ornidyl, MDL 71782, Bey 1978) was a compound of Merrell Dow Research Insti- tute, Strasbourg Center, The syntheses of NI,N4-bis-allenylpu - trescine dihydrochloride (MDL 72527, Bey et al. 1985), DFPut dihy- drochloride (MDL 72720), 6,6DFSpd trihydrochloride (MDL 72766), 7,7-difluorospermidine trihydrochloride (MDL 72748), and 6,6DFSpm tetrahydrochloride (MDL 72968, Gerhart et al. 1987) have been previously published.

Animals and treatment schedules

Adult CD1 albino mice were purchased from Charles River (St. Aubin-les-Elbeuf, France). They were housed in groups of 10 under standardized conditions (22 ~ 60% relative humidity; 12 h light, 12 h dark cycle, free access to standard diet and drinking solution). The drinking solutions were exchanged every 3 days and the amounts consumed per cage and day were noted.

Except controls, which received tap water, animals received a so- lution in tap water containing 3% DFMO and 0.05% MDL 72527 (the PAO inhibitor) for 2 weeks. At an average fluid intake of about 5 ml/day the drug intake corresponded to 4.3 to 5.0 g DFMO and 70 to 80 mg MDL 72527/kg body weight per day (Bolkenius and Seller 1987). On day 15 some animals received 1.27mmo!/kg DFPut (250 mg/kg) (time zero) i.p. Administration of DFMO and MDL 72527 was continued for a further 24 h. From then on all animals received tap water. Groups of mice were killed by decapitation at

certain time intervals. Organs were isolated as quickly as possible, frozen in liquid nitrogen, and stored at - 80 ~ Organs of untreated controls were taken at 24 h and 240 h.

Body weights changed only marginally during treatment; mean values at the time of sacrifice are given in the footnotes to the tables.

Beginning 1 day before DFPut administration 24-h urine samples were collected. Animals were individually housed in stain- less steel metabolic cages and urine was collected in polypropylene tubes containing 0.5 ml of ethanol.

Determination of polyamines and chain-fluorinated polyamines

This was done essentially as described previously by separation of the ion pairs formed with n-octane suifouic acid on a reversed-phase column (Beckman Ultrasphere, I.P.), postcolumn derivatization with o-phthalaldehyde/2-mercaptoethanol, and recording of fluores- cence intensity (Seller and KnSdgen 1985). However, the elution pro- grams were adapted to specific requirements. Column temperature: 22 ~ flow rate 1 ml/min. All changes of solvent composition were linear. For details of equipment, detector reagent etc. see Seller and Kn6dgen 1985.

(a) Determination of difluoropolyamines in tissues and urine:

This elution program is suitable for the establishment of complete polyamine patterns (excluding Nl-acetylputrescine) in tissues, and for the determination of the fluorinated polyamines in tissues and urine. However, the separation of Nl-acetylspermidine and NS-ace - tylspermidine from interfering compounds is not adequate for urine samples.

Solvents: A: 0.1 M sodium acetate (pH 4.50); 10 mM octane sul- fonate. B: 0.2M sodium acetate (pH 4.50)+acetonitrile=4+l; 10 mM octane sulfonate. C: methanol.

Elution time Percent of solvent in min

A B C

0 70 30 0 15 40 60 0 25 20 80 0 30 10 85 5 40 5 85 10 50 0 80 20 75(end time) 0 80 20

(b) Expansion of the region around NLacetylspermidine:

Solvents: A, 0.1 M sodium acetate (pH 4.50); 10 mM octane sulfon- ate. B: 0.2 M sodium acetate (pH 4.50)+acetonitrile+methanol= 100 + 30 + 13; 10 mM octane sulfonate.

Separation was achieved isocratically with a mixture of A + B = 11 + 9; after elution of NLacetylspermidine (26 min) the more polar components (spermidine, spermine etc.) were eluted with eluent B. Equilibration with the initial solvent composition for 8 to 10 min was adequate, before the following sample (200 ~tl in 0.2 M perchloric acid) was applied.

Results

P o l y a m i n e concentrat ions

In Table 1 po lyamine pat terns of non t rea t ed cont ro l animals are given. One group of four mice was ana-

N. Seiler et al.: Chain-fluorinated polyamines as tumor markers. II.

Table 1. Polyamine concentrations in tissues of CD1 albino mice (Mean values + SD; n = 8)

73

Tissue Putrescine N 1-Acetyl- spermidine

Spermidine

nmol per g

N 1 -Acetyl- Spermine Z spermine Polyamines

Brain 10 4- l 1.8-+0.5 Liver 12 4- 3 0.9-+0.4 Kidney 85 4- 24 1.0 4- 0.4 Small intestine 136 4-15 2.5 4- 0.5 Lung 20 4- 3 b.d.1. Heart 11 4- 0.2 b.d.1. Skeletal muscle 9 4- 2 b.d.I. Spleen 81 _4- 6 7 -+2 Skin 21 4- 7 b.d.1.

Adrenals 0.24_+ 0.08 b.d.1.

446 + 24 1140 +186

393 + 33 1352 -+111

383 -+ 46 146 -+ 13

63 -+ 13 1372 4-100

220 __+ 58

nmol per 2 glands 4.5-+ 0.5

b.d.1. 329 + 9 787 + 35 b.d.i. 925 ___84 2078 __+273 b.d.1. 762 +27 1241 _+ 84 b.d.1. 728 __+64 2219 _+190 b.d.1. 353 +37 756 4- 86 b.d.1. 392 -+36 549 + 49 b.d.1. 179 _ 7 251 _4- 22 b.d.1. 1010 -+74 2470 _+182 b.d.1. 127 +32 388 4- 97

b.d.1. 3.5-+ 0.2 8.2_+ 0.8 f

Mean body weight 43 4-1 g b.d.1. =below detectable limits

lyzed at time zero, i.e., at the end of the 15-day treat- ment period, the other 10 days later. Since no signifi- cant difference was observed between the two groups, the mean values were calculated. These values were comparable with previous ones (Bolkenius and Seiler 1987).

The changes in tissues polyamines as observed after 2 weeks treatment with a solution containing 3 % DFMO and 0.05% MDL 72527 instead of drinking water were also comparable with previous results, but the present observations have been extended to 10 dif- ferent organs (Table 2). As a rule, putrescine concen- trations were lowered in all tissues to a level close to or below the detection limit of the method. Spermidine concentrations were usually below 50% of those of controls. Spermine concentrations were either un- changed (brain, heart), or they were significantly ele- vated (liver, kidney, small intestine). In no case were tissue spermine concentrations decreased by the treat- ment with DFMO and MDL 72527.

Owing to the inhibition of PAO, all tissues con- tained measurable concentrations of Nl-acetylsper - midine and Nl-acetylspermine. Highest concentra- tions were found in small intestine and brain. Com- parison of total polyamine concentrations demon- strated an overall diminution of tissue polyamines be- tween 20% (heart) and 33% (small intestine) of con- trols.

The first observable change after termination of treatment was a rapid decline of Nl-acetylspermine concentration, followed by Nl-acetylspermidine. Concomitantly, putrescine concentrations were in- creasing (Table 2).

During the first 24 h after treatment with DFMO and the PAO inhibitor had been stopped, spermidine and spermine concentrations usually started to in-

crease slowly. On day 2 we observed, however, a re- markably rapid increase in spermine levels in several organs, as is exemplified in Fig. 1 for kidney. From then on a gradual decrease in spermine concentration was observed. Although there were considerable dif- ferences between the individual organs, on day 10 nor- mal polyamine patterns had usually been attained. It appears from the data in Tables 2 and 3 and the ex- ample shown in Fig. 1 that the total polyamine pool tended to be higher in some tissues even on day 9 after

15 OO

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d 7" O ID

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~"Polyarnines

Spermine

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DAYS AFTER CESSATION OF TREATMENT

Fig. 1. Replenishment of polyamines in mouse kidney as a function of time after cessation of treatment with D,L-e-difluoromethylorni- thine (DFMO) and the polyamine oxidase (PRO) inhibitor N~,N 4- bis-allenylputrescine (MDL 72527). The dashed lines indicate mean control values. (Mean values + SD)

Table 2. Polyamine concentrations in tissues of mice after 15 days of treatment with DFMO and MDL 72527, and after cessation of ornithine decarboxylase and PAO inactivation (Mean values _+ SD, n = 4)

Tissue Time after Putrescine N1-Acetyl - Sperm• N<Acetyl- Spermine N cessation sperm• spermine Polyamines of treatment days nmol per g

Brain 0 b.d.1. 40 _+ 4 211 _+ 25 3.2 -+ 0.2 345 + 12 599 _+ 41 1 b.d.1. 41 • 3 204 • 14 2.8 _+ 0.5 362 _+ 10 609 _+ 28 2 0.6 _+ 0.1 37 + 7 229 -+ 6 1.1 -+ 0.4 377 _+ 18 644 _+ 32 5 3.1 _+ 1.1 12 -+ 1 299 • 13 b.d.1. 342 + 19 656 -+ 34 9 5.0 _+ 0.8 11 +_ 1 384 _+ 11 b.d.1. 334 • 7 734 _+ 20

Liver 0 2 _+ 1 8.3 _+ 0.2 449 • 92 11.5 _+ 2.0 1039 -+171 1510 +266 1 21 • 8 10.2 -+ 1.4 589 _+ 26 2.6 -+ 1.0 1171 __.118 1794 _+154 2 13 _+ 9 5.6 _+ 1.7 724 _+212 b.d.1. 1185 _+134 1928 __+357 5 11 _+ 5 2.4 -+ 0.3 914 ___179 b.d.1. 1220 __.112 2147 -+296 9 9 _+ 1 1.2 -+ 0.4 952 _+133 b.d.1. 1067 _+ 77 2029 _+211

Kidney 0 2 _+ 2 7.7 _+ 1.9 114 _+ 11 22.2 _+ 1.6 847 ___ 27 993 • 43 I 33 _+ 3 4.7 -+ 0.8 140 -+ 16 2.0 _+ 0.5 813 • 19 993 _+ 39 2 56 _+24 3.7 _+ 0.7 232 _+ 20 2.0 _ 0.7 955 -+ 14 1248 -+ 59 5 69 ___23 2.4 _+ 1.5 350 -+ 31 b.d.1. 905 -+ 33 1326 _+ 89 9 78 _+ 3 1.8 • 1.1 430 • 74 b.d.1. 827 -+ 24 1336 _+103

Smallintestine 0 13 _+ 2 84 +15 613 -+154 15 _+ 5 767 -+ 66 1492 -+242 1 65 ___18 41 _+ 6 857 -+232 2.6 _+ 0.6 923 _+132 1889 ___388 2 185 _+70 16 _+ 9 1171 _+170 b.d.1. 927 _+ 74 2299 +_323 5 97 +34 7 _+ 3 1159 -+ 55 b.d.1. 723 _+ 91 1986 _+183 9 113 +42 5 -+ 2 1181 _+ 75 b.d.1. 685 _+ 56 1984 _+175

Lung 0 1.3 _+ 0.4 8 • 2 152 _+ 40 11 _+ 2 393 • 58 565 _+102 1 10.1 -+ 0.4 6 _+ 2 188 _+ 6 5 _+ 1 450 _+ 41 659 • 50 2 12 _+ 1 8 -+ 2 230 _+ 13 4 _+ 3 433 _+ 39 687 _+ 58 5 21 -t- 6 3.5 -+ 0.5 373 _+ 39 b.d.1. 410 • 30 808 -+ 75 9 15 _+ 3 1.5 _+ 0.6 360 _ 52 b.d.1. 331 • 56 708 _+111

Heart 0 b.d.1. 3.2 _+ 0.6 62 _+ 4 5.0 _+ 0.5 370 • 25 440 _+ 30 1 5 -+ 1 2.8 _+ 0.2 53 -+ 12 2.6 _+ 0.7 396 ___ 34 460 _+ 48 2 7.4 -+ 0.6 2.3 _+ 0.8 82 _+ 19 b.d.1. 411 _+ 73 503 _+ 93 5 8 -+ 2 b.d.1. 108 _+ 18 b.d.1. 360 • 19 476 • 40 9 8.7 _+ 0.7 b.d.1, 133 _+ 9 b.d.1. 367 _+ 23 509 _+ 33

Skeletal muscle 0 b.d.1. 2 _+ 1 22 _+ 6 b.d.1. 186 _+ 8 208 _+ 14 1 2 _+ 0.5 1.8 • 0.7 25 _+ 3 b.d.1. 172 _+ 12 199 _+ 16 2 7 _+ 3 1.5 _+ 0.4 50 _+ 5 b.d.1. 187 • 6 244 _+ 14 5 7 _+ 3 b.d.1. 67 • 21 b.d.1. 178 _+ 8 252 _+ 32 9 8 _+ 2 b.d.1, 94 _+ 12 b.d.1. 171 _+ 8 273 • 22

Spleen 0 22 _+ 8 43 _+ 5 742 _+101 84 _+15 1244 • 86 2135 -+215 1 75 • 45 • 8 934 _+ 45 30 +10 1163 _+ 54 2247 _+127 2 98 -+22 40 _+ 7 1157 -+116 14 -+ 9 1194 _+135 2503 _+189 5 73 _+10 11 _+ 4 1211 _+120 b.d.1. 1045 • 55 2340 _+189 9 67 -+ 6 9 • 3 1285 _+137 b.d.1. 966 _.%_ 72 2327 -+218

Skin 0 4 -I- 2 7 -+ 1 118 _+ 23 b.d.1. 188 -+ 12 317 _+ 38 1 9 _+ 5 5 _+ 1 111 • 8 b.d.1. 167 • 24 292 • 38 2 12 _+12 3 _+ 1 144 -+ 45 b.d.1. 183 • 33 342 _+ 91 5 62 -+29 b.d.1. 294 _+ 60 b.d.1. 174 _+ 26 530 +115 9 49 -+22 b.d.1. 286 _+ 58 b.d.1. 134 _+ 22 469 ___102

Adrenals nmol per 2 glands

0 0.02_+ 0.02 0.08_+ 0.03 2.0+ 0.08 0.03_+ 0.03 4.3_+ 0.4 6.4_+ 0.6 1 0.09+_ 0.03 0.08_+ 0.03 2.3_+ 0.6 b.d.1. 4.4__+ 0.7 6.9_+ 1,4 2 0.19___ 0.06 0.08_+ 0.01 3.3__+ 0.9 b.d.1. 4.0_+ 0.6 7.6_+ 1.6 5 0.20_+ 0.06 0.02_+ 0.02 3.6_+ 0.4 b.d.1. 3.6_+ 0.5 7.4___ 1.0 9 0.21_+ 0.2 b.d.1. 3.7_+ 0.6 b.d.1. 3.5_+ 0.5 7.4_+ 1.1

Groups of four mice received a solution containing 3% DFMO and 0.05% MDL 72527 as sole drinking fluid for 2 tap water. Tissues were sampled between 9 and 10 a.m. Mean body weight (g) day 0: 41.8• h 41.0_+3.2; 2: 40.0+0.8; 5: 40.3+2.2; 9: 41.0_+ 1.8 b.d.1. = below detection limits

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O

Tab

le 3

. (co

ntin

ued)

Tis

sue

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e P

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ter

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mid

ine

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ad

min

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ys

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rmid

ine

NL

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tyl

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rmin

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ut

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FS

pd

6,6D

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pm

Z

Z

sper

min

e P

olya

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es

Dif

luor

o-

poly

amin

es

nmol

per

g

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Adr

enal

s

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11

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16

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48

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3

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10

58

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1.

1 b.

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466

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271

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ceiv

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/kg

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ted

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and

ad

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and

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N. Seller et al.: Chain-fluorinated polyamines as tumor markers. II. 77

subsequently. Except in brain, 6,6DFSpm levels were higher in all tissues containing difluoropolyamines. Maximum concentrations of 6,6DFSpm were ob- served at 48 h, indicating the accumulation of this compound by uptake from the circulation. The major source for the circulating 6,6DFSpm is the small intes- tine, the only tissue with sufficiently high amounts of fluorinated polyamines to cover their observed en- hancement in liver and kidney. This fact is illustrated in Fig. 2. In addition one has to assume 6,6DFSpm formation from 6,6DFSpd.

Tissue spermine levels were either not affected by this treatment (brain, heart, skeletal muscle, adrenals) or they were elevated (liver, kidney, heart, lung, skin, spleen) due to the now well-established fact that pu- trescine and sperm• depletion causes the accumu- lation of decarboxylated S-adenosylmethionine (Ma- mont et al. 1982); in the absence of putrescine only sperm• is available as a substrate for aminopropy- lation reactions, and thus spermine is formed in ex- cess. Consequently, reduction of total polyamine pools in tissues is considerably smaller (20%-30%) than that of sperm• especially since the elevated concentrations of acetylated polyamines also add to the total pool size.

Shortly after cessation of treatment with the two inhibitors Nl-acetylspermine and Nl-acetylsper - re• concentrations decreased, indicating the (par- tial) recovery of PAO, for which an approximate bio- logical half-life of 2-3 days has been estimated (Bolke- nius et al. 1983). Putrescine concentrations increased concomitantly, indicating the rapid recovery of active ODC. The rather steep increase in spermine concen- tration between 24 and 48 h after treatment was stop- ped (Fig. 1) presumably reflects the activation of S- adenosylmethionine decarboxylase by the now ele- vated putrescine concentrations. It is known that ODC inhibitors increase the cellular content of the en- zyme (Persson et al. 1985). It is not known whether an overshoot of active ODC is produced after cessation of DFMO and MDL 72527 administration.

Administration of a single dose of 250 mg/kg (1.27 mmol/kg) of DFPut dihydrochloride had no de- tectable effect on the metabolism of the natural poly- amines, i.e., replenishment of normal polyamine stores was the same in the presence or absence of DFPut, as appears from comparison of the results in Tables 2 and 3. This may be due to the fact that only a relatively small proportion of DFPut was entering the tissues and used as a substrate for sperm• syn-

Table 4. Chain-fluorinated polyamines in mouse urine after administration DFPut All animals received a solution containing 3% DFMO and 0.05% MDL 72527 as sole drinking fluid for 2 weeks. On day 15 the animals were placed individually in metabolic cages. Four mice received 50 mg/kg aminoguanidine sulfate (AG) and all animals 1 h later 1.27 mmol/kg DFPut i.p. Administration of DFMO and MDL 72527 was continued for a further 24 h. From then on four treatment groups (2 animals each) were established: (a) physiological saline; (b) 50 mg/kg AG i.p. daily; (c) MDL 72527 0.05% as drinking fluid; (d) 50 mg/kg AG plus MDL 72527, and 24-h urine samples were collected Values are given in nmol/24 h (mean _+ SD; n=2) (A) Nonhydrolyzed urine (B) Urine hydrolized (6 M HC1, 120 ~ 16 h)

Treatment Time of DFPut 6,6 DFSpd urine collection (A) (B) (A) (B) h

Physiological saline 0-24 17 815 _+ 1025 n.d. 215 • 120 n.d. AG 22035+2623 21053• 348_+ 68 323__+25 MDL 72527 20586• 16640_+2348 437_+ 1 376_+11 AG+MDL72527 22695_+ 804 21648_+ 171 339_+ 27 301•

Physiological saline 24-48 371 • 157 n.d. 132 16 n.d. AG 408• 45 677+ 33 139• 35 144+40 MDL 72527 309+ 243 1458• 78* 154-+ 49 179• A G + M D L 72527 392_+ 291 1708_+ 16" 106_+ 28 125_+25

Physiological saline 48-72 46+ 6 n.d, 47+ 18 n.d. AG 205+ 100 253• 57 49+ 8 30___ 4 MDL 72527 121• 93 384_+ 150 31+ 24 39__+25 A G + M D L 72527 48+ 1 305+ 21" 8+ 3 21__+ 8

An asterisk indicates a statistically significant (P < 0.05) difference between hydrolyzed and non hydrolyzed urine samples (Student's t-test) n.d. = not done

78 N. Sei ler e t al.: C h a i n - f l u o r i n a t e d p o l y a m i n e s as t u m o r marke r s � 9 II .

thase. The major proportion (98% of the fluorinated polyamines that were recovered in urine) was excreted in the form of DFPut; 96% of this amount during the first 24 h, at a time when treatment with the two in- hibitors was continuing, in order to avoid competition of DFPut with the natural substrate of spermidine synthase.

The elimination of fluorinated polyamines from tissues is obviously coupled with the elevation of the levels of endogenous polyamines, as appears from the data of Table 3, and also for kidney from comparison of Fig. 1 and Fig. 2. In general, the steepest decrease of difluoropolyamine analogues was observed at a time, when total polyamines accumulated most rapidly. However, there was no simple quantitative relation- ship between the replenishment of endogenous poly- amine pools, and the elimination of difluoropoly- amine analogues from the various tissues�9

Difluoropolyamines in urine

Of the injected amount of DFPut about 57% could be recovered in urine, as can be calculated from the data in Table 4. Most of it was excreted during the first 24 h. Total recovery of 6,6DFSpd was 1.5%.

In order to evaluate the role of oxidative deamina- tion by copper-containing amine oxidases in the me- tabolism of fluorinated polyamines, 50 mg/kg of ami- noguanidine sulfate was given daily after cessation of the treatment with DFMO and the PAO inactivator. Inhibition of the aminoguanidine-sensitive amine oxi- dases (Seiler et al. 1983) produced a slight elevation of DFPut excretion between 48 and 72 h, but had no sig- nificant effect on 6,6DFSpd excretion�9 If treatment with the PAO inhibitor was continued (or if a combi- nation of the PAO inhibitor and aminoguanidine was administered) this effect was not seen.

Acid hydrolysis of the urine samples decreased somewhat the measurable amounts of DFPut and 6,6DFSpd in the 24 h urines, presumably due to los- ses. However, there was a very considerable increase in the DFPut content in the hydrolysates of the 48 h urines, and also in the samples collected between 48 and 72 h (Table 4). The increase in DFPut content was especially large in the urines of animals treated with MDL 72527 or the combination of MDL 72527 and aminoguanidine. This suggests that a significant amount of the DFPut is excreted in a conjugated form, presumably as the monoacetyl derivative, ex- cept immediately after administration of this com- pound.

In the case of 6,6DFSpd there was a tendency to- ward increased amounts in the hydrolysates of the 48 h and 72 h urines, but the difference between hy- drolyzed and nonhydrolyzed samples was not signifi- cant.

z

= <

�9

t"q

r r ~

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<

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4 1 § 2 4 7 �9 + 1 4 1 + 1 4 1 § 2 4 7 �9 §

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N. Seiler et al.: Chain-fluorinated polyamines as tumor markers. II. 79

In spite of an intense search, 6,6DFSpm could not be detected in any urine sample, nor in urine hydroly- sates.

Continuation of treatment with the PAO inhibitor, or with aminoguanidine sulfate and the PAO inhibitor for 72 h produced the expected increase in N1-ace - tylspermidine and N~-acetylspermine in tissues, but tissue content of chain-fluorinated polyamines was not enhanced by these treatments. On the contrary, 6,6DFSpm contents were usually lower than in the tis- sues of animals without the additional administration of the PAO inhibitor (cf. Tables 3 and 5).

Discussion

After treatment of mice with DFMO and the PAO in- hibitor MDL 72527 for 2 weeks, considerable amounts of Nl-acetylspermidine and Nt-acetylsper - mine were found in all tissues except in skeletal muscle, which contained only Nl-acetylspermidine in measurable quantities. This observation indicates that acetylation of polyamines and degradation of the N 1- acetyl derivatives by PAO are rather general processes in the vertebrate organism. From the depletion of spermidine concentrations by 50% or more in all tissues one may conclude that the reutilization of putrescine that is formed by this catabolic process, for the de novo biosynthesis of spermidine and spermine, is of general physiological significance. Our present findings confirm and extend previous results (Bol- kenius and Seiler 1987; Seiler et al. 1985a, b).

Compared with organs of Lewis lung carcinoma- bearing C57 B1 mice, that were pretreated with DFMO for 5 days (Sarhan et al. 1987) considerably smaller amounts of chain-fluorinated polyamines were found under the present experimental conditions in all tissues of CD1 mice 24 h after a single i.p. dose of 250mg/kg DFPut. However, the ratio of 6,6DFSpm/6,6DFSpd was considerably higher after treatment with the two inhibitors than was found in the previous work, after administration of DFMO alone. The obvious reason is that due to the more complete depletion of the endogenous pool with the inhibitor combination, 6,6DFSpd could more effec- tively compete with spermidine for aminopropylation by spermine synthase.

There are a number of factors involved in the con- trol and limitation of DFPut uptake into tissues and its transformation into 6,6DFSpd and 6,6DFSpm. One of these is most probably the absolute extent of polyamine depletion. Although the relative loss of polyamines is comparable in all tissues, the absolute decrease varies considerably. In small intestine, for ex- ample the loss of spermidine is 750 nmol/g, but in skel- etal muscle only 46 nmol/g. The corresponding figures

for total uptake of fluorinated polyamines in these two extreme cases were 181 nmol/g and 0 nmol/g, respec- tively (Tables 1 and 3). However, the content of fluorinated polyamines in tissues was not directly pro- portional to the total polyamine content, as can be seen from these same tables, suggesting the impor- tance of other factors.

The biological half-life of the aminopropyl moiety of spermidine in mouse liver was estimated to be around 8 days; the putrescine moiety of spermidine apparently had a longer half-life: it varied between 11 days (small intestine) and 16 days (liver, skeletal muscle, kidney), and due to excessive putrescine reutil- ization 42 days in brain (see Antrup and Seiler 1980 for earlier work). The half-life of spermine is presum- ably longer, but no precise data are known for sper- mine turnover rates in tissues.

The rapid elimination of the fluorinated poly- amines, which coincides with the most rapid phase of replenishment of the natural polyamines indicates that the fluorinated polyamines are not following normal patterns of polyamine degradation. Unpublished ob- servations show that they are weak substrates of ace- tylCoA:polyamine Nl-acetyltransferase. Due to the decreased basicity of the amino group in the vicinity of the fluorine atoms, their electrostatic interaction is diminished; therefore, they are displaced by the natu- ral polyamines from anionic binding sites and are sub- sequently eliminated.

Most of the DFPut was rapidly excreted as such. Administration of aminoguanidine, i.e., inhibition of diamine oxidase (E.C. 1.4.3.6.) and related copper- containing amine oxidases (Seiler et al. 1983) pro- duced a slight increase in urinary DFPut excretion (Table 4), suggesting that DFPut may be a (weak) substrate of this type of oxidase.

The fact that administration of the PAO inhibitor caused the enhanced excretion of a DFPut conjugate (Table 4) was unexpected. One potential explanation for this observation is that MDL 72527 induces non- specific acetylation (or another conjugate forming reaction). However, there is no experimental evidence for this type of side effect of the compound. Since monoacetylputrescine is a substrate of monoamine oxidase (MAO, E.C. 1.4.3.4.) (Seiler and A1-Therib 1974) it may also be tempting to speculate that N- monoacetyl-2,2-difluoroputrescine is formed, which is oxidatively deaminated by MAO. However, MDL 72527 is specific for PAO (Bey et al. 1985) hence this assumption is unlikely to be correct.

Most of the 6,6DFSpd in urine was found as such. There is only circumstantial evidence that a small pro- portion of this compound is excreted in the 48 h urine as conjugate, if the PAO inhibitor was administered. An obvious explanation for this finding would be to

80 N. Seiler et al.: Chain-fluorinated polyamines as tumor markers. II.

assume that 6,6DFSpd can to some extent enter the in- terconversion pathway (Seiler et al. 1981) and that degradation of Nl-monoacetyl-6,6-difluorosper - midine by PAO was inhibited. The available observa- tions do not allow us to come to unambigous conclu- sions concerning the metabolism of 6,6DFSpd. Even more puzzling is the metabolic fate of 6,6DFSpm. Al- though it was formed in significant amounts in most tissues, it could not be found as an excretory product, even when the two oxidases which are involved in the major catabolic pathways of the polyamines (Seiler 1987; Seiler et al. 1983, 1985 a) had practically com- pletely been inactivated. Either there is an unknown catabolic pathway in the vertebrate organism, which is capable of transforming 6,6DFSpm into derivatives which were not detected with our methods of urinary- analyses, or 6,6DFSpm or a conjugate of this com- pound is excreted via the bile duct.

Conclusions

Concomitant inactivation of ODC and PAO leads to a more complete depletion of spermidine concentra- tions in all tissues than is possible to achieve with an inhibitor of ODC alone. This suggests that formation of putrescine along the interconversion pathway, and its reutilization for spermidine formation is significant in all vertebrate organs.

In animals with maximally depleted spermidine pools the formation of chain-fluorinated polyamines is significant, but the amounts formed after a 250 mg/ kg dose of DFPut dihydrochloride did not compen- sate for the polyamine losses due to inhibition of pu- trescine formation. Maximum concentrations of total chain-fluorinated polyamines did not exceed 8% of the normal endogenous polyamine pool in small intes- tine and were considerably lower in all other tissues. This favors the use of chain-fluorinated polyamines as markers of tissues with an intense polyamine metabo- lism, since DFPut has been shown to accumulate rather selectively in tumors of DFMO-pretreated ani- mals (Sarhan et al. 1987), in analogy to the accumula- tion of labeled putrescine (Chancy et al. 1983).

The metabolic fate of the chain-fluorinated poly- amines needs further clarification; however, it is al- redy apparent from the present work that they are not interfering significantly with the metabolism of the natural polyamines.

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Received April 1, 1987/Accepted September 17, 1987