Effect of berenil on polyamine metabolism in primary cultured rat hepatocytes

0020-7 I I X/93 $6.00 + 0.00

Int. J. Biochem. Vol. 25, No. 6, pp. 865-868, 1993 Printed in Great Britain. All rights reserved

EFFECT

Copyright 0 1993 Pergamon Press Ltd

OF BERENIL ON POLYAMINE METABOLISM PRIMARY CULTURED RAT HEPATOCYTES S.

Dipartimento

di Medicina

COLOMBATTO

and M. A.

GRILLO

e Oncologia sperimentale, Sezione di Biochimica, Torino 10126, Italy [Fax 01 l-652-77701 (Received

I December

IN

Universita’

di Torino,

1992)

Abstract-l.

Putrescine and spermidine content increased in hepatocytes during culture. In the presence of 10 PM Berenil, putrescine content was further increased, while the increase of spermidine was prevented.

2. Ornithine decarboxylase activity was markedly reduced, and to a lesser extent also S-adenosylmethionine decarboxylase activity. 3. Berenil appears to promote an increase in the transformation of spermidine into putrescine, and to inhibit the polyamine efflux.

INTRODUCTION

72527 was a generous gift from Marion Merrell Dow, Strasbourg. Rat tail tendon collagen was prepared according to Strom er al. (1982). Hepatocyte isolation and culture. Hepatocytes were isolated from adult male rats by a two-step collagenase perfusion procedure (Probst and Unthan-Fechner, 1985). Low centrifugal speeds were used during cell washing to avoid contamination of hepatocytes with non-parenchymal cells. The cells were plated at a density of 7 x IO4 viable cells/cm* on culture dishes coated with rat tail tendon collagen in Ml99 medium supplemented with 2 mg bovine serum albumin, 3.6 mg Hepes, 100 U penicillin, 100 pg streptomycin/ml, 5% horse serum, 0.1 PM dexamethasone and incubated in a humidified incubator with a CO,/air atmosphere (5:95 v/v). Only cell suspensions with a viability (tested by Trypan blue exclusion) of 70% were used. After allowing 4 hr for cell attachment, the medium was changed, except where otherwise specified, to Ml99 medium modified as above, but without horse serum and with 0.5 nM insulin. The medium was changed daily. Assays. After the time chosen the cells were washed with ice cold phosphate saline solution (0.8% NaCI, 0.02% KCI, 0.115% Na*HPO,, 0.02% KH2P04, at pH 7.2) (PBS) and lysed with 20 mM Hepes pH 7.2 containing 0.5% Triton X-100. The supernatant obtained by centrifuging 10 min at 12,OOOg was used to measure ornithine decarboxylase activity (ODC) (Jlnne and Williams-Ashman, 1971) and SAMDC (Sturman, 1976) activity. Polyamines were measured by HPLC in the cells washed as described, extracted with 0.2 M HCIO, and dansylated (Colombatto ef al., 1990). For the efflux assay, hepatocytes were loaded with [ 14C]spermidine (0.025 pCi/ml). After I hr the cells were washed three times with PBS and new medium was added. After 3 hr, part of the medium was used to measure the radioactivity released and part was concentrated under vacuum, dansylated and processed by HPLC as described above, monitoring radioactivity with a Radiomatic Flow one/beta detector (Radiomatic Instruments, Tampa, FL, U.S.A.). Protein was measured according to Lowry et al. (1952).

Several compounds that inhibit S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in the synthesis of spermidine, have been studied. The competitive inhibitor methylglyoxal bis(guanylhydrazone) (MGBG) (Williams-Ashman and Schenone, 1972), an antileukemic drug (Williams-Ashman and Seidenfeld, 1986), has received the greatest attention, followed by a few others (Pegg and WilliamsAshman, 1987). MGBG, however, is not specific and has several other effects. It inhibits diamino oxidase, induces spermidine acetyltransferase and produces severe mitochondrial damage (Pegg, 1986). Attempts to obtain more specific SAMDC inhibitors have therefore continued. A deoxy-adenosine derivative, named MDL 73811, has recently been described (Casara et al., 1989) and its effect on normal and tumor-bearing mice has been extensively studied (Seiler et al., 1991). Another compound described as an irreversible inhibitor of SAMDC is Berenil (4,4’-diamidinodiazoaminobenzene) (Karvonen et al., 1985), a drug widely used in veterinary and human medicine as an antibabesial and antitrypanosomal agent. Its effect on polyamine concentration and on other polyamine metabolism enzymes, however, was not investigated. We report the results of a study of polyamine metabolism and transport in hepatocytes cultured in the presence of Berenil. MATERIAL

AND METHODS

Horse serum, penicillin/streptomycin and Ml99 were obtained from Flow Laboratories (Irvine, U.K.); Berenil, collagenase, bovine serum albumin from Sigma Chemical Co. (St. Louis, MO, U.S.A.); [I-‘4C]ornithine, S-adenosyk[carboxyl-14C]methionine and [1,4-‘4C]spermidine from Amersham (U.K.). The polyamine oxidase inhibitor MDL 865

S. COLOMBATNI and

866 RESULTS

M. A. GRILLO

AND DISCUSSION

I

Hepatocytes were cultured in the presence of IOpM Berenil. After 24-96 hr, cells were collected and used to measure polyamines. As shown in Fig. 1, putrescine content increased during culture, as previously reported (Colombatto and Grille, 1991). In the presence of Berenil, it was accumulated in higher amounts, particularly in the first 24 to 48 hr. In contrast, s~~idine, which also increased in normal cells, did not change significantly in the presence of Berenil. Spermine was not affected in the first

1

ODC

4

0

3

I 6

hr

Fig. 2. Effect of Berenil on ODC and SAMDC activity in

(a) 15

r

hepatocytes in culture. Cells were cultured as described under Methods for 48 hr. Then 10 PM Berenil (final concentration) was added and enzyme activities were measured at the time shown. The results shown are representative of three independent experiments in duplicate. Initial ODC activity (100%) was 10.2 + 0.9 pmol/hr/mg protein Initial SAMDC activity (100%) was 410 f I5 pmol/hr/mg protein.

DAB

24

0

48

72

96

hr

0)

SPD k

Control

l

Berenil

1

I

I

I

24

48

12

96

hr

(cl

SPM

I5 r

I

h

Control

.

Berenil

I

I

I

I

24

48

72

96

hr

Fig. I. Putrescine, spermidine and spennine in hepatocytes cultured in the presence of 10pM Berenil. The results shown are representative of three independent experiments in duplicate.

48 hr, but its subsequent increase in normal cells was prevented. Marked decrease of ODC activity was observed between 3 and 6 hr after administration of Berenil. The effect on SAMDC was less marked and short lasting: after 6 hr, the activity was again almost normal (Fig. 2). The increase in putrescine content, therefore, cannot be attributed to an effect on ODC activity. One possible explanation is activation of the interconversion pathway. To show whether this was the case, polyamines were measured in hepatocytes cultured in the presence of 5 FM Berenil and 20 FM MDL 7257, a specific inhibitor of polyamine oxidase. As shown in Table 1, Berenil at this concentration promoted modifications similar to those shown before. In the presence of the polyamine oxidase inhibitor, N’-acetylspermidine was accumulated and acetylspermine was also detectable. When both compounds were present, the increase of putrescine was prevented and Nra~etyls~rmidine was further accumulated, while spermidine was decreased. These results suggest that Berenil promotes an increase in the interconversion pathway. The lack of modification of acetylspermine content may be due to the higher affinity of polyamine oxidase for acetylspermine (Tsukada, 1988). Apparently the enzyme is not completely inhibited, and the residual activity is able to oxidise acetylspermine, so that it does not accumulate further. These data therefore suggest that Berenil is able to promote synthesis of putrescine from spermidine. While this manuscript was in preparation, Berenil has been shown (Libby and Porter, 1992) to be, like MGBG (Pegg et al., 1985) a competitive inhibitor of SAT. In viva, however, MGBG is able to increase SAT activity, at least in part due to an increase in the half-Iife of the

Berenil and polyamine Table

metabolism

867

I. Polyamine content in rat hepatocytes cultured in the presence of 5 pM Berenil

Addition

DAB

None

2.50 3.48

5 p M Berenil 20pM MDL 72527 5 uM Berenil + MDL 72527

1.57 2.10

N ‘-AcSPD

SPD

nmol/mg protein 0.27 8.26 0.35 7.43

I .53 3.04

5.95 3.93

A&PM

SPM

nd nd 0.52 0.46

2.44 2.63 5.17 4.62

The cells were cultured as described under Methods in the presence or in the absence of 5 FM Berenil and 20 pM MDL 72527, and processed for the assay of polyamines after 48 hr. The results shown are representative of three independent experiments in duplicate. nd = not detectable.

Table 2. Effect of Berenil on the release of radioactivity from hepatocytes loaded with [“Clspermidine Radioactivity (dumlhrima

Addition None 5 p M Berenil IO FM Berenil

released orotein)

1733 747 587

enzyme (Pegg et al., 1985). The same could occur with Berenil, a structural analogue of MGBG. Polyamine transport out of the cell could also alter the polyamine content. To show whether this was the case, hepatocytes were loaded with [ “C]spermidine for 1 hr, then washed and supplemented with fresh medium containing, or not, 5 or 10pM Berenil. The radioactivity released after 3 hr was markedly reduced in the presence of 5 FM Berenil (57%) and even more (66%) with 10pM Berenil (Table 2). Radioactive putrescine and N’-acetylspermidine were found in lower amounts in the medium and in higher amounts in the cells (Table 3). This lends further support to the suggestion that inhibition of the efflux may be responsible, at least in part, for the modification of the cell polyamine content. In conclusion, these results show that inhepatocytes in culture Berenil reduces ODC and (to a lesser extent) SAMDC activity. Nevertheless, putrescine content is increased in the cells, apparently due to interconversion of polyamines and to inhibition of the efflux.

Addition

polyamines hepatocytes DAB

released and retained with (‘4C]spermidine N’-AcSPD dpm/mg

work was supported Scientifica e Tecnologica

by Minister0 (Quote 40%

REFERENCES

Cells were cultured as described under Methods. After 48 hr, they were loaded with [‘4C]spermidine (O.O25pCi/ml) for I hr, then washed 3 times with PBS. Fresh medium was added and Berenil (5 or IOpM final concentration) as required. Radioactivity released was measured after 3 hr. The results shown are representative of three independent experiments in duplicate.

Table 3. Radioactive loading

Acknowledgement-This Universita e Ricerca and 60%).

in 3 hr after SPD

protein

Medium None 5 p M Berenil

2571 1083

I500 360

nd nd

Cells None 5 PM Berenil

9756 12,186

3304 4048

63,452 55,297

Culture conditions as for Table 2. Radioactive polyamines were measured in the cells and in the medium as described under Methods. The results shown are representative of three independent experiments in duplicate. nd = not detectable.

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868

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and M. A. GIULM

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