Carcinogenesis vol.6 no. 12 pp. 1713-1720, 1985

Early stimulation of polyamine biosynthesis during promotion byphenobarbital of diethylnitrosamine-induced rat livercarcinogenesis. The effects of variations of the S-adenosyl-L-methionine cellular pool

Francesco Feo, Renato Garcea, Lucia Daino,Rosa Pascale, Lucia Pirisi, Serenella Frassetto andMaria Emilia Ruggiu

Istituto di Patologia generale dell'University di Sassari, Via P.Manzella 4,07100 Sassari, Italy

A decrease of S-adenosyl-L-methionine liver content wasobserved between the 14th and the 35th day after the startof 2-acetylaminofluorene feeding in diethylnitrosamine-initiated rats according to the 'resistant-hepatocyte' modelof hepatocarcinogenesis. The decrease was enhanced byphenobarbital given to the animals after the end of 2-acetyl-aminofluorene feeding. These changes were associated withan increase in ornithine decarboxylase activity and the spermi-dinerspermine ratio. S-adenosyl-L-methionine administrationto rats caused a great fall in the percentage of 7-glutamyl-transpeptidase-positive liver as well as in polyamine synthesis.An increase in ornithine decarboxylase activity, associatedwith a decrease in the liver S-adenosyl-L-methionine pool, alsooccurred in normal animals on the first day following a par-tial hepatectomy and was enhanced by phenobarbital. Theassociation of 2-acetylaminofluorene feeding with partialhepatectomy resulted in a slower liver regeneration, while thedecrease in S-adenosyl-L-methionine level and the increasein polyamine synthesis were observed over a longer periodof time after partial hepatectomy. These changes were fur-ther prolonged in diethylnitrosamine-initiated rats in which7-glutamyltranspeptidase-positive foci developed. In theseanimals a high level of polyamine synthesis was still presentwhen liver regeneration was complete. At this stage of theobservation period the labeling index was very low in sur-rounding liver, but still high in the 7-glutamyltrans-peptidase-positive areas. Phenobarbital stimulated polyaminesynthesis and cell growth and further prolonged the periodof time during which a high ornithine decarboxylase activityand labeling index were present. These results indicate thatthe liver llpotrope content could be a rate-limiting factor forcell growth and liver neoplasia promotion and this could de-pend on the modulation of polyamine biosynthesis.

IntroductionPolyamine (PA)* biosynthesis plays an essential role in normalas well as neoplastic growth (1—4). A rate-limiting enzyme inPA biosynthesis, ornithine decarboxylase (ODC) increases ear-ly during growth (1—4); growth is inhibited by various inhibitorsof this enzyme (4,5). Different observations indicate that skincarcinogenesis promoters induce ODC (6 - 8) and S-adenosyl-L-methionine (SAM) decarboxylase (6) activities.

Systemic investigations of PA metabolism during liver carcino-

•Abbreviations: 2-AAF, 2-acetylaminofluorene; DENA, diethylnitrosamine;GGT, 7-glutamyltranspeptidase; LI, labeling index; ODC, ornithine decarboxy-lase, PA, polyamine; PB, phenobarbital; PH, partial hepatectomy; SAM, S-adenosyl-L-methionine; SPD, spermidine; SPE, spermine.

genesis are scanty and sometimes debatable. Scalabrino et al.(9) reported a bimodal increase of rat liver ODC and SAM decar-boxylase activities during prolonged 4-dimethylaminoazobenzenefeeding. A first peak of activity was observed 1 month after star-ting the carcinogen feeding, and the second peak 3 months later.However, these experiments did not clarify whether the increaseof ornithine and SAM decarboxylase activities were specificallyinvolved in the neoplastic transformation, or depended on theregenerative process which follows liver damage produced bylong-term carcinogen administration. PA biosynthesis has alsobeen studied during the initiation and promotion phases of multi-step carcinogenesis. No induction of ODC activity was foundin the livers of rats fed a phenobarbital (PB)-containing diet, asa promoting stimulus, after an initiating short-term treatment withdiethylnitrosamine (DENA) (10). In contrast, a slightly increas-ed level of ODC activity was observed by Yanagi et al. (11) inthe livers of rats receiving PB 2 weeks after a single necrogenicDENA dose. Olson and Russell (12) investigated the PA syn-thetic activity of rat liver in die early post-initiation period, aftera single DENA dose, and during the promoting treatment. Thisconsisted of a partial hepatectomy (PH) at the mid-point of a2-week feeding period of a diet containing 2-acetylaminofluorene(2-AAF) (13). An increase in cAMP kinase and ODC activitiesand in the spermidine:spermine (SPD/SPE) ratio followed DENAinjection. After decreasing to the control level in ~ 7 days, theabove parameters were stimulated once again by PH and remain-ed high for 14 days. However, these researches gave no evidencethat the increase of ODC activity ( 1 0 - 12) and SPD/SPE ratio(12) occurred only in the livers of rat subjected to the initiatingDENA injection, before the promoting treatment, and not in thosesubjected to the promoting stimulus alone. In the latter animalspre-neoplastic lesions are scanty or absent (13). Consequently,the effect of the presence of a putative pre-neoplastic populationin the livers of DENA-treated rats, could not be evaluated.Recently, Pereira et al. (14) compared the ability of differentbarbiturates to induce rat liver ODC activity. They found thatthe more potent ODC inducers were the only barbiturates thatenhanced the formation of 7-glutamyltranspeptidase (GGT)-positive foci when adminstered to DENA-treated rats. This couldbe a clue to the existence of a correlation between PA synthesisand growth of putative pre-neoplastic cells.

Different observations correlate the promotion of hepatocar-cinogenesis and ODC activity to the liver lipotrope content. Infact: (i) chronic administration of a lipotrope-deficient dietenhances rat liver carcinogenesis induced by different carcinogens(15,16). Moreover, recent evidence indicates that such a diet,without any added carcinogen and with barely detectable levelsof some carcinogens, is associated with the development ofhepatocarcinomas (17,18); (ii) this treatment leads to an increaseof ODC and SAM decarboxylase activities (19) as well as to adecrease of hepatic SAM content (20); (iii) a decrease of the SAMpool is also caused by long-term feeding of PB (21), a well knownpromoting agent (22 — 24) which induces ODC activity(11,14,25).

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On the basis of these observations it could be suggested thatthe liver SAM pool may regulate liver neoplasia promotionthrough a modulation of PA synthesis and growth. This hypo-thesis has been tested by studying whether the PB-promoting ef-fect is regulated by variations of the liver SAM pool and if suchvariations are involved in the control of PA synthesis, in an ex-perimental system in which no lipotrope-deficient diet is given.

Materials and methodsMale Wistar rats (160— 180 g) were produced in our laboratory and housed in-dividually in plastic shoe cages, in a room kept at constant humidity (70%) andtemperature (22°C) with a 6 a.m. to 8 p.m. photoperiod. They were placed for2 weeks on a standard high casein diet (Piccioni, Brescia, Italy) with water adlibitum, and received a single 200 mg/kg i.p. dose of DENA, at the end of thefirst week. Putative pre-neoplastic foci were produced according to the 'resistant-hepatocyte' model (13) with some minor modifications. In brief, 1 week afterDENA injection, the animals were fed a standard diet containing 0.03% 2-AAF,with a PH performed at the mid-point of this period. At the end of 2-AAF feedingthe rats were fed a basal diet containing, when indicated, 0.05% PB. SAMtreatments were performed by administering to rats 25 mg/kg body wt. of SAMin the form of disulfate p-toluene sulfonate (BioResearch, Liscate, Milano). SAMwas injected i.m. as a solution (20 mg/ml) in 0.2 M Na2PO4 (final pH 5) at 4-hintervals. Controls received the same amount of phosphate brought to pH 5 withan equimolar mixture of sulphuric acid and p-toluene sulpbonic acid. SAM in-jections were started at the end of 2-AAF feeding and continued unul the ratswere killed. T.l.c. analyses (20 x 7 cm silica gel G plates, N-butanol, acetk acid,H2O, 4:1:1 by vol. as solvents) revealed that SAM preparations were free ofmethionine. H.p.l.c. analyses (see below) showed that S-adenosyl-homocysteineand other unidentified contaminants did not exceed 1.9%.

PH was performed under ether anaesthesia by removal of the median and leftlobes comprising 2/3 of the liver (26). The operations were done between 9 a.m.and 10 a.m. PB treatment of the rats subjected to PH alone was performed byinjecting i.p. 80 mg/kg body wt of PB 1 h before the operation. Twenty-fourhours later, the rats were fed a standard diet containing 0.05% PB. SAM (25 mg/kgbody wt) was injected i.m. 1 h before PH and then every 4 h until the rats werekilled.

For histology small pieces of liver, taken from each of the lobes, were frozenin isopentane at — 140°C and cut, after embedding in Paraplast, in a cryostatinto -5-^m thick sections. The sections were fixed in ice-cold acetone. GGTwas detected according to Ruthenberg a al. (27). For the labeling index (LI)determination the rats were given methyl [3H)thymidine i.p. at a dose of 0.5 iiCiigbody wt (78.6 Ci/mmol, New England Nuclear, Dreieich, GFR). The animalsreceived four injections every 6 h before killing. The rats subjected to PH alonewere given only one labeled thymidine dose 1 h before the sacrifice. The sliceswere fixed and processed for GGT histochemistry as above. Thereafter, they werecoated with Kodak NTB2 emulsion, stored overnight in a desiccator at 23°C andimmersed for 10 s in a Beckman NA scintillation fluid. They were then storedfor 24 h at -75°C. After developing, the slices were counterstained with hematox-ylin for microscopic examination. Morphometric analysis of liver sections wasperformed with the aid of a Leitz-T.A.S. analysis system.

To determine ODC activity, PA and SAM contents, rat liver was rapidly per-fused, under light ether anaesthesia, with 20 ml of ice-cold saline, before killingin order to avoid the contamination of intracellular with extracellular SAM, inSAM-treated rats. No influence of the perfusion was observed on ODC activityand PA content. For the assay of ODC activity, liver samples were homogenis-ed using a Polytron PT-10 homogeniser at full speed (three bursts for 5 s each)in 2.5 volumes of 100 mM Tris-Cl buffer (pH 7.2) containing 0.1 mM pyridoxalphosphate and 0.1 mM EDTA. The homogenates were centrifuged 60 min at30 000 g and the supernatants were used as a source of enzyme. ODC activitywas determined by measuring the 14COj released from L[l-"C]ornithine. Thereaction mixture contained, in 0.2 ml: 100 mM Tris-Cl buffer (pH 7.4), 1.5 mMpyridoxal phosphate, 1.5 mM EDTA, 50 mM dithiothreitol, 2 mM L-ornithinecontaining 1 piCi of DL(l-l*C]omithine (53.7 mCi/mmol, New England Nuclear)and amounts of supernatant corresponding to 2 mg of protein. The reaction mix-ture, free of ornithine, was pipetted into Eppendorf tubes. The tubes were plac-ed in scintillation vials on the bottom of which were put, as a CO, trapping agent,0.2 ml of 6 x 103 mM NaOH adsorbed on a 1 cm* piece of paper. 40 1̂ of or-nithine solution were pipetted on the wall of the Eppendorf tubes, ~ 3 mm overthe surface of the reaction mixture. The vials were then closed with rubber capsand, after 5 min of pre-incubation at 37°C, omithine was rapidly mixed withthe reaction medium. The incubation was run for 60 min at 37°C and the reac-tion was stopped by injecting, through the rubber caps, 0.4 ml of 400 mM HC1O4.After an additional 1 h incubation the tubes were removed from the vials. Thelatter were filled with 15 ml of Beckman MP scintillation fluid and shaken for

12 h. The radioactivity was then determined in a Beckman LS 1800 liquid scin-tillation counter.

In order to determine PA, liver samples were homogenised in 2.5 volumesof ice-cold 400 mM HC1O,. The homogenates were centrifuged 15 min at10 000 g. The sediments were suspended in 100 mM KOH, precipitated againby adding 1 volume of HCIO4 and centrifuged. From the collected supernatantsPA were extracted by water-saturated N-butanol according to the method of Kramerel al. (28) and then purified and estimated according to the method of Fleisherand Russel (29). For h.p.l.c. determination of SAM the liver was homogenisedin 2.5 volumes of chilled 400 mM HCIO4. The homogenates were centrifugedand the supernatants were diluted 1:2 (v:v) with 200 mM ammonium formiatebuffer (pH 4). 200 gl of diluted supernatants were injected into the h.p.l.c. (PerkinElmer Series 10) with an 84-S u.v. detector and a Partisil SCX 10 /im column(25 x 0.46 cm i.d.; Whatman Chem. Sep. Ltd., Maidstone, Cleveland). Thecolumn was maintained at room temperature with a constant flow rate of 1 ml/minand eluted with a 20-min gradient of 10 — 40% methanol in 200 mM ammoniumformiate buffer. Effluent was monitored at 254 nm. The retention time of SAMwas 9 min under the above conditions. Liver SAM was determined by compar-ing the area of the peak in the tissue extract with that of a standard solution.Protein was determined as previously reported (30).

ResultsGGT-positive fociThe data in Table I show the appearance of GGT-positive areasafter PH in 2-AAF fed rats. In the animals subjected toDENA/2-AAF/PH, 10 days after the start of 2-AAF feeding,6.2% of the liver was constituted by GGT-positive areas. Thesefurther increased in number and diameter 14 days after the startof 2-AAF feeding. On the 21st day a 42% decrease of liver oc-cupied by GGT-positive foci occurred. This, however, wasfollowed by a progressive increase during the period of obser-vation. On the 49th day, -25% of liver was GGT-positive. SAMtreatment reduced by 29 - 35 % the GGT-positive liver betweenthe 21st and the 49th day after the start of 2-AAF feeding. Thiswas due to a decrease in number and sometimes also in diameterof the GGT-positive foci. The treatment with PB caused an in-crease in diameter, apparently not in number, of GGT-positiveareas which led to, respectively, 73, 78, 48 and 54% increasesin liver occupied by GGT-positive foci on days 21, 35, 42 and49 after the start of 2-AAF feeding. SAM treatment completelyabolished the PB effect by causing a significant decrease in thenumber and diameter of GGT-positive areas.

No GGT-positive foci were found in rat liver between the 14thand the 49th day after initiating 2-AAF feeding, when the pro-moting treatment, with or without PB, was not preceded byDENA injection (not shown).

SAM liver contentThe results in Table II show that 2-AAF alone had no effect onliver SAM content while 2-AAF combined with PH caused a42% decrease 14 days after the start of 2-AAF feeding, the nor-mal SAM content being restored on the 21st day. When the ratswere subjected to DENA/2-AAF/PH, 35, 26, 27 and 24%decreases of SAM content occurred, respectively 14, 21, 35 and42 days after the start of 2-AAF feeding. Control values wererestored by SAM injection. In the rats treated with PB, 47, 40and 39% decreases of liver SAM content were recorded on days21, 35 and 42. The hepatic SAM pool was restored to 84 - 87 %of the control level by SAM treatment.

The decrease in SAM liver content with or without PB wasnot coupled with a decrease in food intake. Food consumptionbetween days 14 and 49 after the start of 2-AAF, was 12.8 ±2.6 g/rat/day (S.D., n = 29) for control rats and 14.2 ±1.6 g/rat,day (S.D., n = 29) for the rats subjected to 2-AAF/PHor DENA/2-AAF/PH. PB and SAM treatments did not modifyfood intake. In the same period of time the body weight gain

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Table I. GGT-positive

Treatments0

areas in carcinogen-treated liver

Timeb

(days)GGT-positive areas0

Number/mm2 DiameterMaximum Minimum

% Liveroccupied byCKj'l + areas

DENA + 2-AAF + PH

DENA + 2-AAF + PH + SAM

DENA + 2-AAF + PH + PB

DENA + 2-AAF + PH + PB + SAM

101421354249

1421354249

21354249

21354249

0.821.371.131.231.221.22

1.270.640.670.800.77

1.171.311.281.40

0.520.700.801.00

±±±±±±

±±±±±

±±±±

±±±±

0.080.050.030.180.020.71

0.070.07d

0.06d

0.02d

O.O^

0.330.040.090.51

0.05e

0.06c

0.03e

O.OS*

0.280.440.040.700.710.91

0.290.430.480.530.49

0.891.121.161.23

0.380.630.580.68

±±±±±±

±±±±±

±±±±

±±±±

0.040.020.060.020.030.60

0.020.050.02d

0.04d

0.06d

0.10*1

0.19^0.12d

0.6811

0.03C

o.irO.OSPO . ^

0.16 ± 0.050.30 ± 0.010.30 ± 0.050.31 ± 0.060.42 ± 0.020.38 ± 0.08

0.24 ± 0.020.25 ± 0.04d

0.29 ± 0.060.26 ± 0.04d

0.34 ± 0.07

0.45 ± 0.14d

0.61 ± 0.14d

0.60 ± 0.04d

0.64 ± 0.26d

0.15 ± 0.04e

0.30 ± O.OSP0.27 ± 0 .0T0.32 ± 0.05c

6.219.510.618.221.225.2

16.47.6

12.416.318.0

23.532.434.138.9

10.414.814.016.6

±±±±±±

±±±±±

±±±±

±±±±

2.31.61.22.21.55.3

2.61.0"2.81.6"3.O1

6.2"

2.2d

5.5d

1.3C

5.6C

2.5*4.r

"The rats received a single i.p. dose (200 mg/kg) of DENA and, 1 week later, a diet containing 0.03% 2-AAF for 14 days, with PH on the 7th day. Whenindicated the rats were given a diet containing 0.05% PB and/or SAM (25 mg/kg, i.m., every 4 h). PB and SAM treatments were started at the end of2-AAF feeding.bPeriod of Ume from the beginning of 2-AAF feeding.cMeans ± S.D. of triplicate determinations with 6 — 9 rats.dDifferent from DENA/2-AAF/PH for at least p <0.05.•"Different from DENA/2-AAF/PH/PB for at least p <0 .01 .

Table n. S-Adenosyl-L-methionine

Treatments

Control liver

2-AAF

2-AAF + PH

DENA + 2-AAF +PH

DENA + 2-AAF +PH + SAM

DENA + 2-AAF +PH + PB

DENA + 2-AAF +PH + PB + SAM

Time"(days)

142135

142135

14213542

14213542

213542

213542

content

SAMb

in carcinogen-treated liver

Oig/g of liver)

(6)21.4

(3) 20.7(3) 20.4(3) 19.3

(2) 12.3(3) 19.6(3) 22.9

(5) 13.9(5) 15.8(6) 15.6(5) 16.3

(6) 23.3(6)21.2(4) 24.8(6) 24.6

(6) 11.3(6) 12.9(3) 13.0

(5) 17.9(4) 18.5(3) 18.4

±

±±±

±±

±±±±

±±±±

±

±

±±±

2.1

1.30.42.4

2.42.4

0.2c

0.6c

0.8*0.6c

0.4d

2.0"4.0"1.2"

0.6cd

0.3 c d

o.r"0.3c

0.9=Q.T

% Differencefrom control

35262724

474039

161313

"Period of lime from the beginning of 2-AAF feeding.""Means ± S.D. In parentheses is the number of experiments.cDifferent from control for p <0.001.""Different from DENA/2-AAF/PH for at least p < 0 . 0 1 .'Different from DENA/2-AAF/PH/PB for p <0.001.

was 2.8 — 3.3 g/rat/day for all rat groups.

Polyamine synthesisIn Table IH the variations of ODC activity in the animals sub-jected to different treatments are illustrated. It appears that whenPH was performed 7 days after the beginning of 2-AAF feedinga sharp increase of ODC activity occurred on the 14th day.Thereafter, ODC activity progressively decreased. On the 21stday after the start of 2-AAF feeding the enzymatic activity was1.8 times higher than control. Control activity was restored onthe 35th day. PB treatment, initiated 14 days after the start of2-AAF feeding, did not alter ODC activity on days 21 and 35(not shown). In the rats treated with DENA/2-AAF/PH therewas an increase of ODC activity, with the highest activity observ-ed on the 14th day. Thereafter, ODC activity progressivelydecreased without reaching the control values. On the 14th day,the enzymatic activity was still 67% higher than in the controlliver. PB further increased ODC activity between days 21 and49. SAM injection caused a great reduction of activity in bothPB-treated and untreated rats. In the latter animals ODC activi-ty decreased to control values on the 49th day as a consequenceof SAM treatment while in PB-treated rats the enzymatic activi-ty, though greatly inhibited by SAM, was higher than controlactivity during the whole period of observation.

A statistical analysis of the results of ODC and SAM deter-mination is reported in Figure 1. Each value of ODC activityfor liver from different sources is plotted against the correspon-ding SAM content. It can be seen that a correlation coefficientof -0.73 may be calculated (n = 16, p < 0.001).

ODC in the presence of in vitro added SAM was studied to

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Table III. Variations of ornithine decarboxylase activity in carcinogen-treated liver

Table IV. Effect of S-adenosyl-L-methionine in vitro on liver ornithinedecarboxylase activity

Treatments Time*(days)

ODC activity1"(pmol 14C<Vh, mgprotein)

Control liver2-AAF

2-AAF +

DENA +

DENA ++ SAM

DENA +

DENA ++ SAM

PH

2-AAF H

2-AAF H

2-AAF H

2-AAF H

h PH

- PH

- PH + PB

- PH + PB

142135

142135

1421283549

21283549

21283549

21283549

(9)

(3)(3)(3)

(7)(6)(6)

(7)(7)(2)(7)(4)

(7)(2)(7)(2)

(6)(2)(6)(3)

(7)(3)(6)(2)

36.4539.5733.3530.36

139.6065.4939.45

121.1282.5774.8369.7250.68

66.5657.8162.9432.10

159.30143.00106.8583.21

70.7365.0067.1666.00

±±±±

±

±-i-

±

±±

±

±

±

±±

5.9918.223 232.07

5.60°7.70°3.44

7.04°13.80°

6.26°11.60°

2.37c-d

4.8Ocd

14.90c-d

15.3O°d

SAF-d

1.99°c

0.24°e

4.20°e

"Periods of time after the beginning of 2-AAF feeding.bMeans ± S.D. In parentheses is the number of experiments.cDifferent from control forp < 0.001.dDifferent from DENA/2-AAF/PH for at least p <0.01.'Different from DENA/2-AAF/PH/PB for at least p <0.01.

_ 200

oa:a. 150

- 100>->I—o

oQO

50

r—0.73

5 10 15 20SAM

25 30

Fig. 1. Scatter diagram with a linear regression showing a negativecorrelation between the SAM liver content and ODC activity. Theregression coefficient is significantly different from zero (p <0.001).

investigate if SAM directly affected this enzymatic activity. Thedata in Table IV show that the presence of 5 mM SAM in thereaction mixture used for the determination of ODC activity waswithout effect. In contrast, a 36.8% inhibition occurred whenliver homogenate was incubated for 1 h with the same SAMamount before isolating the soluble fraction used as a source ofODC. The pre-incubation of this fraction with SAM, beforedetermining the enzyme activity, had no effects.

The variations of ODC activity during in vivo treatment were

Pre-incubation1 SAM in thereactionmixture

ODC activity11

(pmol I4CO2/h, mg ofprotein)

HomogenateHomogenate + SAM (5 mM)CytosolCytosol + SAM (5 mM)

None5 mMb

None1.25 mMc

None1.25 mMc

34.1832.7332.2220.3728.9928.02

±

±±

±

3.42

1.481.09s

2.36

"Homogenate or cytosol were pre-incubated 60 rrun at 37"C. At the end ofthe incubation the homogenate was centrifuged in order to isolate thecytosolic fraction. This fraction was used as a source of ODC activity.bAddcd to the reaction mixture at zero time.cCalculated on the basis of the SAM presumably added with the cytosol tothe reaction mixture at the zero time.dMeans of two experiments or means ± S.D. of five experiments.cDifferent from pre-incubated homogenate for p <0.001.

paralleled by modifications of PA content and SPD/SPE ratio(Table V). 2-AAF feeding did not modify the PA content but,when PH was performed, an increase in putrescine and SPD oc-curred 14 days after the start of 2-AAF feeding. This led to a5.5-fold increase^ the SPD/SPE ratio. Thereafter, putrescineand SPD underwent a great decrease though not to the controlvalues. A small increase of SPE was also recorded on days 21and 35, so that no changes in the SPD/SPE ratio took place inthis period of time. PB treatment did not modify this behavior(not shown). A single dose of DENA, followed by 2-AAF andPH, led to a large increase in putrescine and SPD between days14 and 35 after the start of 2-AAF feeding. A small increasein SPE also occurred. However, a great rise in SPD caused theSPD/SPE ratio to increase 2.3—4 times. This ratio decreasedto the control values on the 49th day. Analogous changes wereobserved between the 21st and the 49th day after the start of2-AAF feeding, in PB-treated rats. In these animals the highestvalues of putrescine and SPD contents were found on the 21stand 35th days, while on the 49th day the content of the two PAsdecreased though not to the appropriate control. As a consequenceof these changes the SPD/SPE ratio increased between the 21stand the 49th day in respect of that found in the rats subjectedto DENA/2-AAF/PH. SAM injection of PB-treated rats causeda significant decrease in the putrescine content, except on the21st day when an increase was recorded. Moreover, a 42 — 54%decrease of the SPD content, in both PB-treated and untreatedrats occurred. In contrast, the SPE content was not affected orunderwent very small changes. Consequently a great decreaseof the SPD/SPE ratio occurred. This ratio returned to controlvalues on the 49th day in the absence of PB and on the 35th inits presence.

Liver growth in carcinogen-treated ratsThe effects of PB and SAM on the LI and the relative weightof carcinogen-treated liver, after PH, are illustrated in Table VI.It appears that 14 days after the start of 2-AAF feeding, the LIwas 2.5 times higher in GGT-positive foci than in surroundingliver. At this time a 52% liver regeneration occurred. Thereafterthe LI decreased progressively in both GGT-positive and sur-rounding liver. However, it remained higher in GGT-positivefoci than in surrounding liver during the period of observation.The relative liver weight further increased after the 14th day andliver regeneration was complete on the 21st day. As a conse-

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Table V. Variations of the polyamine content in carcinogen-treated liver

Treatments Time"(days)

Putrcscine

nmol/mg protein

0.02 ± 0.00

0.03 ± 0.000.02

1.290.05 ± 0.03d

0.080.14 ± 0.03d

0.57 ± 0.03d

0.27 ± 0.05d

0.11 ± 0.05d

0.02 ± 0.00

0.500.18 ± 0.02e

0.04 ± 0.02c

0.03 ± 0.01

0.23 ± 0.050.22 ± 0.06e

0.18 ± 0.01c

0.50 ± 0.10f

0 080.07 ± 0.05f

Spermidine(A)

4.5 ± 0.6

5.5 ± 0.46.0

21.96.8 ± 1.6"6.08.1 ± l. ld

28.8 ± 1.6d

22.8 ± 4.1d

15.7 ± 4.1d

4.5 ± 1.9

15.99.3 ± 0.2c

8.1 ± 0.3e

4.6 ± 2.0

22.7 ± 2.123.6 ± 4.5C

8.0 ± 2.0c

13.2 ± 1.5f

8.44.4 ± 1.9f

Spermine(B)

4.4 ± 0.8

4.5 ± 1.43.6

4.05.5 ± 0.5"1

3.66.4 ± O.^

6.2 ± 0.3d

5.7 ± 0.1d

6.0 ± 1.2d

3.6 ± 1.2

5.84.7 ± 0.3c

5.9 ± 1.54.4 ± 2.3

4.2 ± 0.35.9 ± 0.8*4.3 ± 2.0c

4.4 ± 0.46.83.7 ± 1.6

A/B°

Control liver

2-AAF

2-AAF + PH

DENA + 2-AAF + PH

DENA + 2-AAF + PH + SAM

DENA + 2-AAF + PH + PB

DENA + 2-AAF + PH + PB + SAM

14

21

14

21

35

49

14

21

35

49

14

21

35

49

21

35

49

21

35

49

CS.D. of the spermidineispermine ratio = ± A/B°y AJ B2

dDifferent from control liver for at least p <0.01.'Different from DENA/2-AAF/PH for at least p <0.05.fDifferent from DENA/2-AAF/PH/PB for at least p <0.05.

0.20.4

0.3

101.21.2

5.51.2 ±1.61.3 ± 0.23.3 ± 1.2*4.0 ± 0.7"*2.3 ± 0.8"1.2 ± 0.62.7

1.9 ± 0.1c

1.4 ± 0.3c

1.0 ± 0.75.4 ± 0.44.0 ± 0.5e

1.9 ± 0.3e

3.0 ± 0.4f

1.2

1.2 ± 0.7f

•Penod of time from the beginning of 2-AAF feeding.bMeans ± S.D. of triplicate determinations with 3 — 9 rats or means of triplicate determinations with 2 rats.

Table VI. Effect of phenobarbital and S-adenosyl-L-methionine on

Treatments

DENA + 2-AAF+ PH

DENA + 2-AAF +PH + SAM

DENA + 2-AAF +PH + PB

DENA + 2-AAF +PH + PB + SAM

Days after starting14

Labeling1

indexGGT +

8.86C

±1.62

SAT*±0.58

i

GGT-

3.53±0.25

1 1.63d

±0.38

2-AAF

Liver"weightg/lOOg

2 1±0.1

1.6d

±0.4

feeding

%

52±2.3

3 9 d

±1.8

18Labeling"indexGGT +

8.00^±1.02

1.26cd

±0.01

16.02cd

±1.44

4.22CX

±1.24

the labeling index

GGT-

2.74±0.04

0.24d

±0.00

3.55d

±0.00

1.20*±0.00

Liver"weightg/100g

2.5±0.3

2.0±0.02

4.4± 0 8

2.8±0.4

and liver weight

%

62±2.0

45^±1.6

100±4.0

75*±1.4

21Labeling"indexGGT+

6.01c

±0.90

0.95^^±0.00

9.68cd

±1.28

3.16"±1.18

in carcinogen-treated rats

GGT-

0.25±0.00

0.16d

±0.00

0.25±0.00

0.30±0.03

Liver"weightg/lOOg

4.0±0.2

3.8±0.5

4.5±0.5

4.5±0.9

%

100±2.3

92d

±1.9

100±1.2

100±2.4

42

Labeling"indexGGT+

1.48°±0.21

1.15°±0.12

1.44C

±0.04

LOT±0.14

G G T -

0.18±0.18

0.19±0.01

0.26±0.05

0.2±0.03

Liver"weightg/100g

4.0±0.4

4.1±0.1

4.4±0.7

4.4±0.5

%

100±1.7100±1.6100±2.0100±1.6

T h e rats were given four i.p. injections of methyl [3H]thymidine (0.5 iiCi/g body wt), every 6 h before killing. The LI was determined by counting 8000hepatocytes per liver in 100— 140 GGT-positive areas (GGT+) and in surrounding (GGT-) . Data are means ± S.D. of triplicate determinations with 4 — 6animals."Calculated as relative weight (g/100 g body wt) or as percentage of the average weight found in each rat group the 49th day, when no further increaseoccurred.dDifferent from G G T - for at least p <0.01.""Different from DENA/2-AAF/PH for at least p <0.01.eDifferent from DENA/2-AAF/PH/PB for p <0.001.

quence of SAM treatment, a large decrease in the LI in GGT-positive islands and in surrounding liver was observed, whileliver regeneration proceeded more slowly than in untreated

animals. In PB-treated rats the LI of GGT-positive foci increas-ed markedly, in respect to that of untreated rats, 18 and 21 daysafter the start of 2-AAF feeding. Although some increase of the

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Fig. 2. Effect of PB and SAM on the liver relative weight, ODC activity,LI and SAM content in hepatectomized rats. PB (80 mg/kg body weight)was injected i.p. to rats 1 h before PH. 24 h later the rats were fed astandard diet containing 0.05% PB. When indicated, SAM (25 mg/kg bodyweight) was injected i.m. 1 h before PH and then every 4 h until the ratswere killed. To determine the LI, the rats were given an i.m. injection ofmethyll'HJthymidine (0.5 jiCi/kg body weight), 1 h before lulling. Themean LI was determined from 10 000 cells/liver and is expressed as % ofnuclei containing at least four granules. Data are means ± S.D. of 4 - 7experiments. Control values (rats subjected to sham operation) for ODC:38.42 ± 3.82 pmol "CO, released/h, mg protein; LI: 0.7 ± 0.09%; SAMcontent: 24.3 ± 1.02 pg/g. symbols are: (D), without PB; ( • ) , withoutPB, plus SAM; (O), with PB; ( • ) , with PB, plus SAM. V-test: relativewt: PB-treated versus untreated, p <0.005 at days 2 and 4; PB plus SAM,p <0.001 at days 2, 4 and 7. ODC activity: PB-treated versus untreated,p <0.005 between days 0.125 and 4; without PB plus SAM versus withoutPB, at least p <0.01 at days 1 and 2; PB plus SAM versus PB, p <0.05between days 0.5 and 4. LI: PB-treated versus untreated, at least p <0.05between days 1 and 4; without PB plus SAM versus without PB, p <0.05between days 1 and 4. PB plus SAM versus PB, at least p <0.05 betweendays 0.5 and 4. SAM content: PB-treated versus untreated, p < 0.005 atdays 1 and 2; without PB plus SAM versus without PB, p <0.001 at dayI; PB plus SAM versus PB, p <0.001 between days 1 and 4.

LI also occurred in surrounding liver on day 18, on the 18th and21st days the LI was respectively, 3.5 and 10 times higher inthe GGT-positive population than in the surrounding hepatocytes.PB also stimulated liver regeneration which was complete 18 daysafter the start of 2-AAF feeding. Again, SAM treatment induceda significant decease of LI in both GGT-positive and surround-ing liver, and caused liver regeneration to proceed more slowly.

Studies with regenerating liverIn order to assess whether the variations of SAM pool, PA syn-thesis and growth depended on the PH, the SAM content, ODCactivity and LI were studied in rats subjected to PH alone. Thedata in Figure 2 indicate that after PH the relative liver weightincreased faster in PB-treated rats. The regeneration was com-plete after 4 days in these rats, while in untreated animals the

relative weight increased to 95 % of the maximum values 7 daysafter PH, these values being reached on the 14th day. After thistime no further increase of liver weight occurred (not shown).In the rats not treated wth PB, SAM did not significantly inhibitliver regeneration, while it significantly inhibited it between the1st and the 7th day after PH in PB-treated animals. ODC activ-ity exhibited a very large increase after PH, with two peaks at4 and 12 h which were, respectively, 1.2 and 1.8 times higherin PB-treated than in untreated rats. Thereafter, the enzymaticactivity progressively decreased, the control values being reachedafter 4 days in the absence, and 7 days in the presence, of PB.There was a small inhibition of ODC activity by SAM between0.5 and 2 days after PH. However, in PB-treated rats, a 2- to2.3-fold inhibition was observed between 0.5 and 4 days. TheLI progressively increased after PH reaching a peak 24 h afterPH in PB-treated and untreated rats. Thereafter, it decreased inall rat groups, reaching the control values 4 — 6 days after PH.The decrease was slower in PB-treated animals which still ex-hibited a relatively high LI 2 and 3 days after PH. In the presenceof SAM the LI was greatly inhibited and fell to normal values3 days after PH. As concerns the liver SAM content it can beseen in Figure 2 that one day after PH a 70% and a 44% decreaseoccurred in PB-treated and untreated rats, respectively. SAM con-tent reached the control values the 2nd day after PH, in theanimals not subjected to PB, and the 3rd day in those treatedwith PB. SAM decrease was prevented by exogenous SAM in-jection.

Discussion

Our data show that liver ODC activity and PA synthesis inhepatectomised rats is largely influenced by carcinogen ad-ministration. As already observed (31), when no carcinogens areadministered, a bimodal increase of ODC activity occurs witha first peak 4 h, and a second peak 12 h, after PH. Then, ODCactivity progressively decreases reaching in 4 days the valuesfound in non-hepatectomised rats. As already seen (32) liverregeneration proceeds faster in PB-treated rats. This is also shownby the fact that 2 and 3 days after PH, the LI is still high in PB-treated animals, whereas it is low in untreated rats. This behavioris coupled to a slower decrease of ODC activity which, in thepresence of PB, reaches the control values 2 days later than inits absence. It thus seems that the stimulation of PA synthesisis an early event in hepatectomised rats not subjected to car-cinogen injection. It precedes the peak of thymidine incorpora-tion into the DNA and is followed by a rapid decrease to thecontrol values in less than one week. In contrast, when PH isassociated with 2-AAF feeding, in the rats not subjected to aninitiating DENA injection, ODC activity is still high 2 weeks afterPH, probably because of the retarding effect of 2-AAF on livergrowth (13). This behavior is apparently not influenced by PB.It should be noted, however, that PB treatment was initiated inthese animals one week after PH so that the effects of the pro-moter on the early post-hepatectomy period could not be ap-preciated. DENA injection preceding the promoting treatment(2-AAF plus PH, with/without PB), further prolongs the increaseof ODC activity and SPD/SPE ratio, a suggested marker of cellgrowth (1), which remains high for at least 4 — 5 weeks after PH.This is particularly evident in PB-treated rats which exhibited,on the 49th day after the start of 2-AAF feeding, an ODC ac-tivity 128% higher than that of control liver. This behavior coulddepend on the presence of a growing putative pre-neoplasticpopulation in the liver of rats subjected to the complete treat-

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Effect of S-adenosyl-L-methfonine on pfaenobarbhal promotion

ment (initiation plus promotion), and could indicate that thispopulation contributes to the elevation of ODC activity. It couldbe suggested that PA synthesis is a necessary event for thedevelopment of putative pre-neoplastic foci. Consistent with thisconclusion is the observation that a high ODC activity andSPD/SPE ratio is present in the rats subjected to DENA/2-AAF/PH/PB 21 days after the start of 2-AAF feeding, when liverregeneration is almost complete and the LI is very low in sur-rounding liver, being still high in GGT-positive areas. Moreover,the inhibition of PA synthesis by SAM is associated with adecrease of GGT-positive liver for at least 49 days after initiating2-AAF feeding.

Our data indicate that variations in the SAM pool may greatlyinfluence regenerative liver growth and the promotion process.This process, whether stimulated or not by PB, is characterisedby a significant decrease in SAM liver content. This agrees withprevious results indicating that prolonged hepatocarcinogen ad-ministration decreases the liver SAM levels (33). The SAMdecrease was observed in this study using the 'resistant-hepatocyte' model of hepatocarcinogenesis promotion (13) whichis not based on the administration of choline and methionine defi-cient diets. On the other hand, no differences in the food intakewere observed between control and experimental animals whichcould explain the decrease of liver SAM content. It could be sug-gested that a decrease of liver SAM content is not specific ofthe promotion induced by lipotrope-deficient diets (34). SAMappears to decrease in growing tissues and this decrease is main-tained for a long period of time when putative pre-neoplastic tissueis growing in the liver. It is reasonable to assume that growingtissues, either normal or pre-neoplastic, need relatively highamounts of SAM for nucleic acid and phospholipid synthesis (35).PB stimulates the transmethylase pathway for phosphatidylcholinesynthesis (36), induces membrane hyperplasia (37) and growthin rat liver, which should further decrease the liver SAM pool.It could be hypothesised that a SAM decrease in growing liverwith a high PA synthesis, further stimulates this synthesis andgrowth. Exogenously administered SAM rapidly enters liver cells(38) apparently without undergoing a preliminary splitting tosmaller molecules (39). Exogenous SAM administration leadsto a reconstitution of the liver SAM pool in the rats subjectedto DENA/2-AAF/PH and to a sharp increase of liver SAM,though not to the control values, in PB-treated rats. Thisphenomenon results in a decrease of cell growth, PA synthesisand putative pre-neoplastic foci formation. Thus, a low SAMpool is associated with PA synthesis and growth. An increaseof ODC activity has been described in the rats fed a choline-deficient diet (19) which is shown to greatly decrease the liverSAM content (20). The existence of a statistical correlation bet-ween liver SAM content and ODC activity clearly indicates thatSAM inhibits ODC activity, but it says nothing about themechanism of this inhibition. The fact that SAM inhibits cytosolicODC activity in vitro, only after pre-incubation with liverhomogenate could indicate that a SAM metabolite, not formedby the post-microsomal supernatant, is responsible for the in-hibitory effect. Further work is in progress to clarify this point.

The mechanism of the PB-promoting effect has not yet beencompletely elucidated. The 'resistant-hepatocyte' model ofhepatocarcinogenesis (13) is based on a selective stimulation ofrare resistant hepatocytes to proliferate in the presence of 2-AAF,which inhibits growth of uninitiated hepatocytes. The brief growthstimulation given by PH, has a strong promoting effect (40). Afterrelease of 2-AAF feeding uninitated cells undergo a rapid pro-liferation which leads to liver regeneration, while many putative

pre-neoplastic foci remodel to form normal appearing liver (41).It has been suggested that PB inhibits the remodeling process(24,42—44). However, our data show that PB highly stimulatesearly liver foci growth. This appears to play a role in the pro-gressive enlargement of the GGT-positive areas, which could in-dicate that remodeling inhibition by PB is not the only mechanisminvolved in the increase of putative pre-neoplastic liver. Perhaps,the maintenance of a proliferative stimulus, represented by PB,over a relatively long period of time contributes to maintain thestability of putative pre-neoplastic cells, which results in aremodeling inhibition.

It is important to note that some enzymatic markers (45) andgrowth (46) of putative pre-neoplastic liver undergo the same,even if sometimes quantitatively different (45,47), adaptiveresponses to PB as normal liver does. According to our obser-vations the PB mitogenic stimulus on the putative pre-neoplasticpopulation is higher and persists for a longer time than that onthe surrounding liver. This could mean that the population ofselected hepatocytes overexpresses some adaptive responses, suchas growth, to the promoter (see also reference 48). Thus, cellgrowth appears to be a very important mechanism, but probablynot the only one, in the PB enhancement of the promotion phasein the 'resistant-hepatocyte' model of hepatocarcinogenesis. Ourresults show that this PB effect is at least partially linked to amodulation of the liver lipotrope content.

AcknowledgementsThis work was supported by grants from the 'Progetto Finalizzato Oncologia'of CNR, and 'Ministero Pubblica Istruzione' (programs 40% and 60%).

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Received on 15 April 1985; accepted on 9 September 1985

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