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Deoxyuridine suppression test on isolated rat bone marrow cells and the in vitro effect of bidisomide
Toxic. in Vitro Vol. 8, No. 2, pp. 277-282, 1994
~
Pergamon
0887-2333(93)E0007-3
ElsevierScienceLtd. Printedin Great Britain 0887-2333/94$7.00+ 0.00
D E O X Y U R I D I N E SUPPRESSION TEST ON ISOLATED RAT BONE MARROW CELLS A N D THE IN VITRO EFFECT OF BIDISOMIDE A. ROGISTER,Y. VANDENBERGHE*and J. ROBA Product Safety Europe, Department of Toxicology, Searle European Development Centre, 11 rue Granbonpr6, 1348 Mont-Saint-Guilbert, Belgium (Received 8 February 1993; revisions received 27 May 1993)
Abstract--The deoxyuridine suppression test was performed on isolated rat bone marrow cells in order to study the effect of bidisomide, a new Class I antiarrhythmic agent, on folate-dependent DNA synthesis. Methotrexate and 5-fluorouracil, two known inhibitors of DNA synthesis, were included in the study to validate the test system. Methotrexate and 5-fluorouracil, at a concentration of 5.5/~M, decreased thymidine incorporation into DNA by way of the de novo pathway (thymidylate synthase activity). salvage pathway of DNA synthesis (thymidine kinase activity), however, was not affected by these anticancer drugs. Bidisomideup to 1 mu did not affect the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells.
The
INTRODUCTION Bidisomide is a new Class I antiarrhythmic agent (Desai et al., 1992) currently in the clinical phase of development. During the chronic administration to rats of bidisomide admixed to feed, treatment-related decreases in reticulocyte (35%), red blood cell (4%) and lymphocyte (20%) counts and an increase in mean corpuscular volume (10%) were observed. A possible functional folate and/or vitamin Bt2 deficiency was suspected from measuring increased formiminoglutamic acid and methylmalonic acid levels in the urine of bidisomide-treated rats. Further evidence for a deficiency was the reversibility of decreased reticulocyte counts and increased formiminoglutamic acid urinary excretion when rats treated with bidisomide received supplement of subcutaneous folinic acid (unpublished results). Folate and/or vitamin B~2 deficiencies can be responsible for an altered DNA synthesis owing to an insufficient deoxythymidylate synthesis (thymine precursor) (Wickramasinghe and Longland, 1974). This synthesis is inhibited by some anticancer drugs, in particular 5-fluorouracil (5-FU) and methotrexate (MTX). 5-FU is known to be an inhibitor of thymidylate synthase activity, and MTX is an inhibitor of the enzyme dihydrofolate reductase (Douglas, 1987) (Fig. 1). The deoxyuridine (dU) suppression test on isolated human bone marrow cells is used routinely as a diag-
*To whom correspondence should be addressed. IdR = thymidine; dU = deoxyuridine; 5Fu = 5-fluorouracil; MTX = methotrexate; THF = tetrahydrofolate.
Abbreviations:
nostic test to detect altered DNA synthesis resulting from folate and/or vitamin B12 deficiencies (Wickramasinghe and Matthews, 1988). The test measures: (A) the incorporation of [3H]thymidine ([3H]TdR) into DNA by way of the salvage pathway catalysed by thymidine kinase, and (B) the inhibitory effect of dU on [3H]TdR incorporation into DNA consecutive to the formation of deoxythymidine monophosphate (deoxythymidylate), a folate-dependent thymidylate synthase catalysed reaction (Fig. 2) (Wickramasinghe and Matthews, 1988). Results are expressed as [3H]TdR incorporation into DNA and as a percentage of suppression of [3H]TdR incorporation into DNA by dU (dU%). Folate antagonists would be expected to cause an increase in the dU% value. The dU suppression test can be performed in the presence of potential correction factors, such as formyl tetrahydrofolate (THF) or folinic acid, 5-methyl THF, pteroylglutamic acid or folic acid and cyanocobalamin (Das and Herbert, 1989; Wickramasinghe and Matthews, 1988). The ability of one or more of these factors to correct altered dU% values may help to determine the mechanism of inhibition of thymidylate synthase activity. The objective of the present study was to evaluate, by use of the dU suppression test, a potential direct or indirect effect of bidisomide on folatedependent thymidylate synthase activity. The test was performed on suspensions of isolated rat bone marrow cells to which the test compound was added for l-hr incubations in a concentration range 1 mM tO 1 riM. Prior to testing the effect of bidisomide, the dU test was validated by the use of 5-FU and MTX.
277
278
A. ROGISTERet al. MATERIALS AND METHODS
Chemicals. Bidisomide, ( +__)-~ [2-[acetyl(l-methylethyl)amino]ethyl]- ~- (2- chlorophenyl) - 1- piperidinebutanamide (mol. wt 407.98) (Fig. 3), was supplied by G. D. Searle & Co. (Skokie, IL, USA). MTX (Ledertrexate SP, 5 mg/2ml, Lederle, Cyanamid, Belgium) and 5-FU (Fluoro-Uracil, 250 mg/10 ml, Roche, Belgium) were obtained from a local pharmacy. [3H]TdR (sp. act. 46 Ci/mmol) was purchased from Amersham (Aylesbury, Bucks., UK). dU, folic acid, folinic acid, 5-methyl THF and cyanocobalamin were obtained from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals were from reputable commercial sources and of the highest available purity. Preparation o f rat bone marrow cell suspension.
Male Sprague-Dawley CD rats aged between 6 and 10 wk were used. Animals were killed by exsanguination under deep anaesthesia (60 mg sodium pentobarbital/kg body weight, ip). Femurs were used for the preparation of the bone marrow cell suspensions. They were cleaned of surrounding muscle tissue. Both the proximal and distal ends were sliced off at the epiphysial plates. Tris-buffered Hanks' solution, pH 7.4, containing preservative-free heparin (25 U/ml) was flushed through the lumen and the marrow was gently forced out of the femur into a collection vessel.
deoxyuridine [
! .....
^ ....... i * ':)"Luum~ Uum~'ktmLm
t
thymidylate synthase
dUMP
/
(deoxyuridylatc)/ formylTHF
The collected marrow was then passed through a 26-gauge needle in order to break up clumps of cells. Cells were resuspended in a known volume of Trisbuffered Hanks' solution. A nucleated cell count was performed by using a Technicon H. 1 cell counter. The cell suspension was finally diluted to a concentration of 3 x 106 nucleated ceils/ml. Deoxyuridine suppression test. Isolated bone marrow cells (final concentration in reaction mixture: 2.7 × 106 nucleated cells/ml) were pre-incubated under constant shaking for 1 hr at 37°C with the test compounds: 5-FU (55 10 J2M-55#M), MTX (55 10-12 M 55 gM) or bidisomide (1 riM-1 mi). When tested, correction factors were added 15 rain prior to the start of the reaction. The following concentrations of correction factors were tested: 5-methylTHF, pteroylglutamic acid (folic acid) and formyl-THF (folinic acid) each at 0.01, 0.1 and 1 mM, and cyanocobalamin at 0.74, 7.4 and 74/~M. The reaction was started by adding [3H]TdR (10 mCi/ml, 10/d/ml incubation medium) to one set of test-tubes and [3H]TdR + dU (final concentration 0.1 mM) to a second set of tubes. All assays were performed in duplicate. The reaction was maintained for 1 hr at 37°C under constant shaking. The reaction was stopped by putting the test-tubes on ice. Cells were washed twice in ice-cold phosphate buffered saline.
J
r
~
=- d T M P
~
(deoxythymidylate)
~
ptero " "
",7
dihydrofolate
5,10- methylene THF ~ . . glycin~
serine
ic acid
/\
~hydrofolate
T. t
"-!
"
(u)trahydrofolam) I Eo'r]I~I£XA~,!,I ~.~-methionine
methyl B 12
~' z-
methioninesynthetase homocysteine
5-meth, ,ITHF
Fig. 1. 5,10-Methylene tetrahydrofolate regenerating cycle in the de novo pathway of deoxythymidylate synthesis. *Methotrexate is an inhibitor of the enzyme dihydrofolate reductase and 5-fluorouracil of thymidylate synthase.
279
dU test: in vitro effect of bidisomide dATP
dGTP
dCTP
(DN~
~
dTI'P
' =
dTMP
thymidine kinase
thymidylate synthase
dMP
ideoxyu6dine
dUDP THYMIDINE
SALVAGE
DE NOVO PATHWAY
PATHWAY Fig. 2. Schematicpresentation of the de novo and salvage pathways of deoxythymidylate(dTMP) synthesis and incorporation into DNA. dATP = deoxyadenosine triphosphate; dCTP = deoxycytidine triphosphate; dGTP = deoxyguanosine triphosphate; dTMP = deoxythymidinemonophosphate; dTTP = deoxythymidine triphosphate; dUDP -- deoxyuridine diphosphate; dUMP = deoxyuridine monophosphate.
Proteins and DNA were precipitated by adding 2ml 0.5M perchloric acid. Supernatant was removed after centrifugation (2000 g, 5 min), and DNA was extracted from the pellet with 0.5 u perchloric acid during a 20-min incubation at 80°C under constant shaking. [3H]TdR incorporated into DNA was counted using a liquid scintillation counter (Wallac 1410, Pharmacia). Results are expressed as dU%, which is an indication of the capacity of dU to inhibit [3H]TdR incorporation into DNA:
CI
O:,. ~
NH 2 CH 3
© Fig. 3. Structure of bidisomide.
dU% = [3H]TdR into DNA in presence of dU (cpm) [3H]TdR into DNA (cpm)
x 100%
RESULTS Effect o f methotrexate and 5-fluorouracil on D N A synthesis in isolated bone marrow cells Tables 1 and 2 show the in vitro effect of MTX and 5-FU on [3H]TdR incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value. Table 3 shows the effect of cofactors in correcting dU% values altered by MTX. TdR values give an indication of the amount of TdR incorporation into DNA by way of salvage pathway (Fig. 2). The conversion of TdR to deoxythymidine monophosphate is catalysed by thymidine kinase. Results in Table 1 indicate that TdR incorporation into DNA by this pathway was not significantly different from the control value when bone marrow cells were incubated with MTX concentrations ranging from 55 10 12M to 55~M. However, when the test was performed in the presence of dU, measuring the activity of thymidylate
A. ROGISTER et al.
280
Table 1. In vitro effect of methotrexate (MTX) on [SH]thymidine(~H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value MTX concn
[3H]TdR _+SD? dU% -+ SD
Control 1297 _+287 9.6±0.7
55 10-J2M 1058 _+242 9.8_+1.7
5.5 nM 1128 ± 177 9.8_+ 1.1
0.55 tiM 1087 _+ 156 14.4_+3.3
55 ~M 1260 _+212 52.6_+3.0***
?[SH]TdR values are expressed in counts per minute. Values represent the mean _+SD of three independent experiments. Asterisks indicate significant differences from control values (***P < 0.001, paired t-test). Table 2. In vitro effect of 5-fluorouracil (5-FU) on [3H]thymidine(3H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value 5-FU concn
[3H]TdR ± SDt
Control
55 10-*2M
5.5 nM
0.55/~M
55/~M
1410 _+ 425
1309 ± 209
1223 _+ 348
1237 ± 310
1468 _+ 405
dU% + SD 9.7-+0.8 9.3-+1.6 9.5 -+ 1.9 12.7-+2.7 39.6_+3.3*** ?[3H]TdR values are expressed in counts per minute. Values represent the mean ± SD of four independent experiments. Asterisks indicate significant differences from control values (***P < 0.001, paired t-test).
s y n t h a s e , M T X at 55//M significantly i n c r e a s e d the d U % value. A l t h o u g h n o t significant, the increase o f the d U % value was a l r e a d y e v i d e n t at 0.55/~M. In a n o t h e r e x p e r i m e n t , M T X was fairly active in inc r e a s i n g the d U % value at 5.5/~M (Table 3). A b n o r m a l d U % values c a u s e d by M T X were c o r r e c t e d by formyi T H F a n d 5 - m e t h y l T H F . D N A synthesis was p a r t l y c o r r e c t e d at 0. l mM a n d c o m p l e t e l y r e s t o r e d at 1.0 mM. P t e r o y l g l u t a m i c acid, 0 . 0 1 - 1 . 0 r a M (results n o t s h o w n ) , a n d c y a n o c o b a l a m i n , 0.74-74/zM, d i d n o t c o r r e c t a n altered D N A synthesis c a u s e d by MTX.
T a b l e 2 s h o w s that 5 - F U , as M T X , in a c o n c e n t r a t i o n r a n g e o f 55 10-12M to 55/~m did n o t affect T d R i n c o r p o r a t i o n into D N A by w a y o f the salvage p a t h w a y , i n d i c a t i n g t h a t 5 - F U h a s n o effect o n t h y m i d i n e kinase activity. A c o n c e n t r a t i o n - d e p e n d e n t increase o f the d U % value was o b s e r v e d b e t w e e n 5.5nM a n d 5 5 # M , i n d i c a t i n g that 5 - F U affects D N A synthesis t h r o u g h the d e n o v o p a t h w a y . N o n e o f t h e c o r r e c t i o n factors tested, p t e r o y l g l u t a m i c acid, 5-methyl T H F , f o r m y l T H F o r c y a n o c o b a l a m i n , were able to c o r r e c t a b n o r m a l d U % values c a u s e d by 5.5/~M 5 - F U (results n o t s h o w n ) .
Table 3. Effect of 5-methyltetrahydrofolatc (methyl THF), formyl THF and cyanocobalamin in correcting dU% values altered by methotrexate (MTX) at 5.5 ~M Correction factor 0.01 dU% -+ SD
dU% -+ SD
0.1
48.2_+6.3
formyl THF (mM) 51.7_+5.6 30.5_+8.0*
48.0_+2.4
0.01 0.1 +methyl THF (raM) 48.4_+7.2 23.3_+6.9**
1.0 12.8_+0.9"* 1.0 15.7_+5.0"**
0.74 7.4 74 + cyanocobalamin (pM) dU% -+ SD 52.1 ±3.9 55.3+3.9 48.1 _+4.5 47.6_+7.9 The values for each correction factor tested represent mean + SD of three (four for methyl THF) independent experiments. Asterisks indicate significant differences from control values (*P <0.05; **P <0.01; ***P < 0.001, paired t-test). Table 4. In vitro effect of bidisomide on [3H]thymidine (3H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value Bidisomide concn Control 1 nM 0.1 ].~M 10,UM I mM [3H]TdR-J-SDt 823_+253 720+249 714+276 840+357 770_+270 dU% ±SD 9.1_+2.0 9.9±2.1 9.8_+1.0 8.5_+1.3 9.0_+0.9 "['[3HJTdR values are expressed in counts per minute. Values represent the mean + SD of four independent experiments.
dU test: in vitro effect of bidisomide Effect o f bidisomide on D N A synthesis in isolated bone marrow cells
Table 4 shows the in vitro effect of bidisomide on [3H]TdR incorporation into D N A of isolated rat bone marrow cells and the effect on the d U % value. In the range 1 nM to I mM, bidisomide had no effect on thymidine kinase activity (TdR values) or on the d U % value. During the same experiments, isolated bone marrow cells were also incubated with bidisomide and the different correction factors. These factors had no effect on the de novo pathway of D N A synthesis in the presence of bidisomide (results not shown).
DISCUSSION
The dU suppression test has proved to be a sensitive biochemical indicator for the detection of folate and/or vitamin Bl2 deficiencies (Wickramasinghe, 1981). The test, since its original procedure (Killmann, 1964), has been modified to be suitable as a routine diagnostic test, mainly used in humans (Wickramasinghe and Matthews, 1988). Although the test is performed preferably on isolated bone marrow cells, attempts were made to develop an assay on isolated peripheral blood lymphocyte cultures stimulated with phytohaemagglutinin (Das and Herbert, 1978) or even on whole blood lymphocyte cultures (Das et al., 1980). However, more recent studies (Matthews and Wickramasinghe, 1988) have indicated that under these conditions the test has limited value as a diagnostic of folate deficiency of the stimulated lymphocytes. For this reason it was decided in the present study to perform the test on bone marrow cells isolated from rat femurs. The dU suppression test measures the reduction by dU of [3H]TdR incorporation into D N A through the salvage pathway (Fig. 2). This, indirectly, gives an indication of the activity of the de novo pathway in synthesizing deoxythymidylate (Fig. 1). In our test conditions, 0.1 mM dU was determined to produce approximately 90% inhibition of [3H]TdR incorporation into the D N A of rat bone marrow cells. MTX and 5-FU increased the d U % value significantly at concentrations above 0.55#~!. In the absence of an effect on thymidine kinase activity, these results indicate an inhibition of folate-dependent deoxythymidylate synthesis. Indeed, both anticancer drugs are known inhibitors of the thymidylate synthesis cycle (Douglas, 1987). MTX is known (Douglas, 1987) to bind tightly, but reversibly, to the enzyme dihydrofolate reductase, causing a decreased synthesis of deoxythymidylate as a result of decreased availability of the cofactor 5,10-methylene T H F (Fig. I). 5-FU inhibits the activity of thymidylate synthase through the formation of the metabolite fluorodeoxyuridine monophosphate, which binds tightly, but reversibly, to the synthase enzyme.
281
It has previously been shown that the addition of vitamin B12 and folate analogues to the assay mixture may enhance [3H]TdR incorporation into D N A o1 folate and/or vitamin B12 deficient cells in a manner specific for the appropriate vitamin deficiency (Taheri et al., 1982). In general, pteroylglutamic acid (folic acid) and formyl T H F (folinic acid) correct the defect in both folate- and cobalamin-deficient cells, while 5-methyl T H F and cyanocobalamin are effective in folate and vitamin Bl2 deficiencies, respectively (Deacon et al., 1980; Van Der Weyden, 1979; Wickramasinghe, 1981; Wickramasinghe and Longland, 1974; Zittoun et al., 1978). The present study tested the ability of pteroylglutamic acid, formyl THF, 5methyl T H F and cyanocobalamin to correct abnormal d U % values caused by MTX and 5-FU. Formyl T H F and 5-methyl THF, but not cyanocobalamin and pteroylglutamic acid (results not shown), were able to correct abnormal d U % values caused by MTX. None of the correction factors tested was able to correct abnormal d U % values caused by 5-FU (results not shown). This confirms that MTX-related changes result from a folate deficiency, while 5-FU inhibits directly the thymidylate synthase activity. The dU suppression test, having been validated in-house with MTX and 5-FU, was used later to study the effect of bidisomide. Results from a recent 1-yr oral toxicity study by dietary inclusion (A. Rogister, unpublished report) suggested a potential compound-related folate and/or vitamin B~2 insufficiency in the rat. Mean morning bidisomide plasma concentrations in this study ranged from 0.2 to 14.5/~g/ml at treatment levels of 90-900 mg/kg body weight/day. These concentrations exceed largely the therapeutic range. The aim of the present study was to determine whether this potential vitamin insufficiency could affect folate-dependent DNA synthesis. The results indicate that concentrations of bidisomide in the range 1 nM to 1 mM did not affect TdR incorporation into D N A of rat bone marrow cells incubated with and without exogenous dU. This indicates that neither the folate-dependent, nor the folate-independent D N A synthesis of rat bone marrow cells is affected by bidisomide treatment in vitro. Addition of cofactors to the incubation medium did not alter the TdR incorporation (results not shown). It was concluded from these experiments that bidisomide at concentrations up to I raM, tested in vitro, does not affect directly or indirectly the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells. REFERENCES
Das K. C. and Herbert V. (1978) The lymphocyte as a marker of past nutritional status: persistence of abnormal lymphocyte deoxyuridine suppression test and chromosomes in patients with past deficiencyof folate and vitamin B12. British Journal of Haematology 38, 219-233.
282
A. ROGtSTER et al.
Das K. C. and Herbert V. (1989) In vitro DNA synthesis by megaloblastic bone marrow: effect of folates and cobalamins on thymidine incorporation and de novo thymidylate synthesis. American Journal of Hematology 31, 11-20. Das K. C., Manusselis C. and Herbert V. (1980) Simplifying lymphocyte culture and the deoxyuridine suppression test by using whole blood (0.1 ml) instead of separated lymphocytes. British Journal of Haematology 26, 72 77. Deacon R., Chanarin I., Perry J. and Lumb M. (1980) Marrow cells from patients with pernicious anaemia cannot use tetrahydrofolate normally. British Journal of Haematology 46, 523 528. Desai B. N., Cook C. S., Claypool W. D. and Garthwaite S. M. (1992) Bidisomide: Class I antiarrhythmic. Drugs of the Future 17, 374-376. Douglas K. T. (1987) The thymidylate synthesis cycle and anticancer drugs. Medicinal Research Reviews 7, 441-475. Killmann S. A. (1964) Effect of deoxyuridine on incorporation of tritiated thymidine: difference between normoblasts and megaloblasts. Acta Medica Scandinavica 175, 483-488. Matthews J. H. and Wickramasinghe S. N. (1988) The deoxyuridine suppression test performed on phyto-
haemagglutinin-stimulated peripheral blood lymphocytes fails to reflect in vivo vitamin BI2 or folate deficiency. European Journal of Haematology 40, 174-180. Taheri M. R., Wickramasinghe R. G., Jackson B. F. A. and Hoffbrand A. V. (1982) The effect of folate analogues and vitamin B12 on provision of thymidine nucleotides for DNA synthesis in megaloblastic anemia. Blood 59, 634-640. Van Der Weyden (1979) Deoxyuridine metabolism in human megaloblastic marrow cells. Scandinavian Journal of Haematology 23, 37-42. Wickramasinghe S. N. (1981) The deoxyuridine suppression test: a review of its clinical and research applications. Clinical and Laboratory Haematology 3, 1-18. Wickramasinghe S. N. and Longland J. E. (1974) Assessment of deoxyuridine suppression test in diagnosis of vitamin BI2 or folate deficiency. British Medical Journal 3, 148-150. Wickramasinghe S. N. and Matthews J. H. (1988) Deoxyuridine suppression: biochemical basis and diagnostic applications. Blood Reviews 2, 168-177. Zittoun J., Marquet J. and Zittoun R. (1978) Effect of folate and cobalamin compounds on the deoxyuridine suppression test in vitamin B12 and folate deficiency. Blood 51,
119-128.
~
Pergamon
0887-2333(93)E0007-3
ElsevierScienceLtd. Printedin Great Britain 0887-2333/94$7.00+ 0.00
D E O X Y U R I D I N E SUPPRESSION TEST ON ISOLATED RAT BONE MARROW CELLS A N D THE IN VITRO EFFECT OF BIDISOMIDE A. ROGISTER,Y. VANDENBERGHE*and J. ROBA Product Safety Europe, Department of Toxicology, Searle European Development Centre, 11 rue Granbonpr6, 1348 Mont-Saint-Guilbert, Belgium (Received 8 February 1993; revisions received 27 May 1993)
Abstract--The deoxyuridine suppression test was performed on isolated rat bone marrow cells in order to study the effect of bidisomide, a new Class I antiarrhythmic agent, on folate-dependent DNA synthesis. Methotrexate and 5-fluorouracil, two known inhibitors of DNA synthesis, were included in the study to validate the test system. Methotrexate and 5-fluorouracil, at a concentration of 5.5/~M, decreased thymidine incorporation into DNA by way of the de novo pathway (thymidylate synthase activity). salvage pathway of DNA synthesis (thymidine kinase activity), however, was not affected by these anticancer drugs. Bidisomideup to 1 mu did not affect the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells.
The
INTRODUCTION Bidisomide is a new Class I antiarrhythmic agent (Desai et al., 1992) currently in the clinical phase of development. During the chronic administration to rats of bidisomide admixed to feed, treatment-related decreases in reticulocyte (35%), red blood cell (4%) and lymphocyte (20%) counts and an increase in mean corpuscular volume (10%) were observed. A possible functional folate and/or vitamin Bt2 deficiency was suspected from measuring increased formiminoglutamic acid and methylmalonic acid levels in the urine of bidisomide-treated rats. Further evidence for a deficiency was the reversibility of decreased reticulocyte counts and increased formiminoglutamic acid urinary excretion when rats treated with bidisomide received supplement of subcutaneous folinic acid (unpublished results). Folate and/or vitamin B~2 deficiencies can be responsible for an altered DNA synthesis owing to an insufficient deoxythymidylate synthesis (thymine precursor) (Wickramasinghe and Longland, 1974). This synthesis is inhibited by some anticancer drugs, in particular 5-fluorouracil (5-FU) and methotrexate (MTX). 5-FU is known to be an inhibitor of thymidylate synthase activity, and MTX is an inhibitor of the enzyme dihydrofolate reductase (Douglas, 1987) (Fig. 1). The deoxyuridine (dU) suppression test on isolated human bone marrow cells is used routinely as a diag-
*To whom correspondence should be addressed. IdR = thymidine; dU = deoxyuridine; 5Fu = 5-fluorouracil; MTX = methotrexate; THF = tetrahydrofolate.
Abbreviations:
nostic test to detect altered DNA synthesis resulting from folate and/or vitamin B12 deficiencies (Wickramasinghe and Matthews, 1988). The test measures: (A) the incorporation of [3H]thymidine ([3H]TdR) into DNA by way of the salvage pathway catalysed by thymidine kinase, and (B) the inhibitory effect of dU on [3H]TdR incorporation into DNA consecutive to the formation of deoxythymidine monophosphate (deoxythymidylate), a folate-dependent thymidylate synthase catalysed reaction (Fig. 2) (Wickramasinghe and Matthews, 1988). Results are expressed as [3H]TdR incorporation into DNA and as a percentage of suppression of [3H]TdR incorporation into DNA by dU (dU%). Folate antagonists would be expected to cause an increase in the dU% value. The dU suppression test can be performed in the presence of potential correction factors, such as formyl tetrahydrofolate (THF) or folinic acid, 5-methyl THF, pteroylglutamic acid or folic acid and cyanocobalamin (Das and Herbert, 1989; Wickramasinghe and Matthews, 1988). The ability of one or more of these factors to correct altered dU% values may help to determine the mechanism of inhibition of thymidylate synthase activity. The objective of the present study was to evaluate, by use of the dU suppression test, a potential direct or indirect effect of bidisomide on folatedependent thymidylate synthase activity. The test was performed on suspensions of isolated rat bone marrow cells to which the test compound was added for l-hr incubations in a concentration range 1 mM tO 1 riM. Prior to testing the effect of bidisomide, the dU test was validated by the use of 5-FU and MTX.
277
278
A. ROGISTERet al. MATERIALS AND METHODS
Chemicals. Bidisomide, ( +__)-~ [2-[acetyl(l-methylethyl)amino]ethyl]- ~- (2- chlorophenyl) - 1- piperidinebutanamide (mol. wt 407.98) (Fig. 3), was supplied by G. D. Searle & Co. (Skokie, IL, USA). MTX (Ledertrexate SP, 5 mg/2ml, Lederle, Cyanamid, Belgium) and 5-FU (Fluoro-Uracil, 250 mg/10 ml, Roche, Belgium) were obtained from a local pharmacy. [3H]TdR (sp. act. 46 Ci/mmol) was purchased from Amersham (Aylesbury, Bucks., UK). dU, folic acid, folinic acid, 5-methyl THF and cyanocobalamin were obtained from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals were from reputable commercial sources and of the highest available purity. Preparation o f rat bone marrow cell suspension.
Male Sprague-Dawley CD rats aged between 6 and 10 wk were used. Animals were killed by exsanguination under deep anaesthesia (60 mg sodium pentobarbital/kg body weight, ip). Femurs were used for the preparation of the bone marrow cell suspensions. They were cleaned of surrounding muscle tissue. Both the proximal and distal ends were sliced off at the epiphysial plates. Tris-buffered Hanks' solution, pH 7.4, containing preservative-free heparin (25 U/ml) was flushed through the lumen and the marrow was gently forced out of the femur into a collection vessel.
deoxyuridine [
! .....
^ ....... i * ':)"Luum~ Uum~'ktmLm
t
thymidylate synthase
dUMP
/
(deoxyuridylatc)/ formylTHF
The collected marrow was then passed through a 26-gauge needle in order to break up clumps of cells. Cells were resuspended in a known volume of Trisbuffered Hanks' solution. A nucleated cell count was performed by using a Technicon H. 1 cell counter. The cell suspension was finally diluted to a concentration of 3 x 106 nucleated ceils/ml. Deoxyuridine suppression test. Isolated bone marrow cells (final concentration in reaction mixture: 2.7 × 106 nucleated cells/ml) were pre-incubated under constant shaking for 1 hr at 37°C with the test compounds: 5-FU (55 10 J2M-55#M), MTX (55 10-12 M 55 gM) or bidisomide (1 riM-1 mi). When tested, correction factors were added 15 rain prior to the start of the reaction. The following concentrations of correction factors were tested: 5-methylTHF, pteroylglutamic acid (folic acid) and formyl-THF (folinic acid) each at 0.01, 0.1 and 1 mM, and cyanocobalamin at 0.74, 7.4 and 74/~M. The reaction was started by adding [3H]TdR (10 mCi/ml, 10/d/ml incubation medium) to one set of test-tubes and [3H]TdR + dU (final concentration 0.1 mM) to a second set of tubes. All assays were performed in duplicate. The reaction was maintained for 1 hr at 37°C under constant shaking. The reaction was stopped by putting the test-tubes on ice. Cells were washed twice in ice-cold phosphate buffered saline.
J
r
~
=- d T M P
~
(deoxythymidylate)
~
ptero " "
",7
dihydrofolate
5,10- methylene THF ~ . . glycin~
serine
ic acid
/\
~hydrofolate
T. t
"-!
"
(u)trahydrofolam) I Eo'r]I~I£XA~,!,I ~.~-methionine
methyl B 12
~' z-
methioninesynthetase homocysteine
5-meth, ,ITHF
Fig. 1. 5,10-Methylene tetrahydrofolate regenerating cycle in the de novo pathway of deoxythymidylate synthesis. *Methotrexate is an inhibitor of the enzyme dihydrofolate reductase and 5-fluorouracil of thymidylate synthase.
279
dU test: in vitro effect of bidisomide dATP
dGTP
dCTP
(DN~
~
dTI'P
' =
dTMP
thymidine kinase
thymidylate synthase
dMP
ideoxyu6dine
dUDP THYMIDINE
SALVAGE
DE NOVO PATHWAY
PATHWAY Fig. 2. Schematicpresentation of the de novo and salvage pathways of deoxythymidylate(dTMP) synthesis and incorporation into DNA. dATP = deoxyadenosine triphosphate; dCTP = deoxycytidine triphosphate; dGTP = deoxyguanosine triphosphate; dTMP = deoxythymidinemonophosphate; dTTP = deoxythymidine triphosphate; dUDP -- deoxyuridine diphosphate; dUMP = deoxyuridine monophosphate.
Proteins and DNA were precipitated by adding 2ml 0.5M perchloric acid. Supernatant was removed after centrifugation (2000 g, 5 min), and DNA was extracted from the pellet with 0.5 u perchloric acid during a 20-min incubation at 80°C under constant shaking. [3H]TdR incorporated into DNA was counted using a liquid scintillation counter (Wallac 1410, Pharmacia). Results are expressed as dU%, which is an indication of the capacity of dU to inhibit [3H]TdR incorporation into DNA:
CI
O:,. ~
NH 2 CH 3
© Fig. 3. Structure of bidisomide.
dU% = [3H]TdR into DNA in presence of dU (cpm) [3H]TdR into DNA (cpm)
x 100%
RESULTS Effect o f methotrexate and 5-fluorouracil on D N A synthesis in isolated bone marrow cells Tables 1 and 2 show the in vitro effect of MTX and 5-FU on [3H]TdR incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value. Table 3 shows the effect of cofactors in correcting dU% values altered by MTX. TdR values give an indication of the amount of TdR incorporation into DNA by way of salvage pathway (Fig. 2). The conversion of TdR to deoxythymidine monophosphate is catalysed by thymidine kinase. Results in Table 1 indicate that TdR incorporation into DNA by this pathway was not significantly different from the control value when bone marrow cells were incubated with MTX concentrations ranging from 55 10 12M to 55~M. However, when the test was performed in the presence of dU, measuring the activity of thymidylate
A. ROGISTER et al.
280
Table 1. In vitro effect of methotrexate (MTX) on [SH]thymidine(~H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value MTX concn
[3H]TdR _+SD? dU% -+ SD
Control 1297 _+287 9.6±0.7
55 10-J2M 1058 _+242 9.8_+1.7
5.5 nM 1128 ± 177 9.8_+ 1.1
0.55 tiM 1087 _+ 156 14.4_+3.3
55 ~M 1260 _+212 52.6_+3.0***
?[SH]TdR values are expressed in counts per minute. Values represent the mean _+SD of three independent experiments. Asterisks indicate significant differences from control values (***P < 0.001, paired t-test). Table 2. In vitro effect of 5-fluorouracil (5-FU) on [3H]thymidine(3H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value 5-FU concn
[3H]TdR ± SDt
Control
55 10-*2M
5.5 nM
0.55/~M
55/~M
1410 _+ 425
1309 ± 209
1223 _+ 348
1237 ± 310
1468 _+ 405
dU% + SD 9.7-+0.8 9.3-+1.6 9.5 -+ 1.9 12.7-+2.7 39.6_+3.3*** ?[3H]TdR values are expressed in counts per minute. Values represent the mean ± SD of four independent experiments. Asterisks indicate significant differences from control values (***P < 0.001, paired t-test).
s y n t h a s e , M T X at 55//M significantly i n c r e a s e d the d U % value. A l t h o u g h n o t significant, the increase o f the d U % value was a l r e a d y e v i d e n t at 0.55/~M. In a n o t h e r e x p e r i m e n t , M T X was fairly active in inc r e a s i n g the d U % value at 5.5/~M (Table 3). A b n o r m a l d U % values c a u s e d by M T X were c o r r e c t e d by formyi T H F a n d 5 - m e t h y l T H F . D N A synthesis was p a r t l y c o r r e c t e d at 0. l mM a n d c o m p l e t e l y r e s t o r e d at 1.0 mM. P t e r o y l g l u t a m i c acid, 0 . 0 1 - 1 . 0 r a M (results n o t s h o w n ) , a n d c y a n o c o b a l a m i n , 0.74-74/zM, d i d n o t c o r r e c t a n altered D N A synthesis c a u s e d by MTX.
T a b l e 2 s h o w s that 5 - F U , as M T X , in a c o n c e n t r a t i o n r a n g e o f 55 10-12M to 55/~m did n o t affect T d R i n c o r p o r a t i o n into D N A by w a y o f the salvage p a t h w a y , i n d i c a t i n g t h a t 5 - F U h a s n o effect o n t h y m i d i n e kinase activity. A c o n c e n t r a t i o n - d e p e n d e n t increase o f the d U % value was o b s e r v e d b e t w e e n 5.5nM a n d 5 5 # M , i n d i c a t i n g that 5 - F U affects D N A synthesis t h r o u g h the d e n o v o p a t h w a y . N o n e o f t h e c o r r e c t i o n factors tested, p t e r o y l g l u t a m i c acid, 5-methyl T H F , f o r m y l T H F o r c y a n o c o b a l a m i n , were able to c o r r e c t a b n o r m a l d U % values c a u s e d by 5.5/~M 5 - F U (results n o t s h o w n ) .
Table 3. Effect of 5-methyltetrahydrofolatc (methyl THF), formyl THF and cyanocobalamin in correcting dU% values altered by methotrexate (MTX) at 5.5 ~M Correction factor 0.01 dU% -+ SD
dU% -+ SD
0.1
48.2_+6.3
formyl THF (mM) 51.7_+5.6 30.5_+8.0*
48.0_+2.4
0.01 0.1 +methyl THF (raM) 48.4_+7.2 23.3_+6.9**
1.0 12.8_+0.9"* 1.0 15.7_+5.0"**
0.74 7.4 74 + cyanocobalamin (pM) dU% -+ SD 52.1 ±3.9 55.3+3.9 48.1 _+4.5 47.6_+7.9 The values for each correction factor tested represent mean + SD of three (four for methyl THF) independent experiments. Asterisks indicate significant differences from control values (*P <0.05; **P <0.01; ***P < 0.001, paired t-test). Table 4. In vitro effect of bidisomide on [3H]thymidine (3H-TdR) incorporation into DNA of isolated rat bone marrow cells and the effect on the dU% value Bidisomide concn Control 1 nM 0.1 ].~M 10,UM I mM [3H]TdR-J-SDt 823_+253 720+249 714+276 840+357 770_+270 dU% ±SD 9.1_+2.0 9.9±2.1 9.8_+1.0 8.5_+1.3 9.0_+0.9 "['[3HJTdR values are expressed in counts per minute. Values represent the mean + SD of four independent experiments.
dU test: in vitro effect of bidisomide Effect o f bidisomide on D N A synthesis in isolated bone marrow cells
Table 4 shows the in vitro effect of bidisomide on [3H]TdR incorporation into D N A of isolated rat bone marrow cells and the effect on the d U % value. In the range 1 nM to I mM, bidisomide had no effect on thymidine kinase activity (TdR values) or on the d U % value. During the same experiments, isolated bone marrow cells were also incubated with bidisomide and the different correction factors. These factors had no effect on the de novo pathway of D N A synthesis in the presence of bidisomide (results not shown).
DISCUSSION
The dU suppression test has proved to be a sensitive biochemical indicator for the detection of folate and/or vitamin Bl2 deficiencies (Wickramasinghe, 1981). The test, since its original procedure (Killmann, 1964), has been modified to be suitable as a routine diagnostic test, mainly used in humans (Wickramasinghe and Matthews, 1988). Although the test is performed preferably on isolated bone marrow cells, attempts were made to develop an assay on isolated peripheral blood lymphocyte cultures stimulated with phytohaemagglutinin (Das and Herbert, 1978) or even on whole blood lymphocyte cultures (Das et al., 1980). However, more recent studies (Matthews and Wickramasinghe, 1988) have indicated that under these conditions the test has limited value as a diagnostic of folate deficiency of the stimulated lymphocytes. For this reason it was decided in the present study to perform the test on bone marrow cells isolated from rat femurs. The dU suppression test measures the reduction by dU of [3H]TdR incorporation into D N A through the salvage pathway (Fig. 2). This, indirectly, gives an indication of the activity of the de novo pathway in synthesizing deoxythymidylate (Fig. 1). In our test conditions, 0.1 mM dU was determined to produce approximately 90% inhibition of [3H]TdR incorporation into the D N A of rat bone marrow cells. MTX and 5-FU increased the d U % value significantly at concentrations above 0.55#~!. In the absence of an effect on thymidine kinase activity, these results indicate an inhibition of folate-dependent deoxythymidylate synthesis. Indeed, both anticancer drugs are known inhibitors of the thymidylate synthesis cycle (Douglas, 1987). MTX is known (Douglas, 1987) to bind tightly, but reversibly, to the enzyme dihydrofolate reductase, causing a decreased synthesis of deoxythymidylate as a result of decreased availability of the cofactor 5,10-methylene T H F (Fig. I). 5-FU inhibits the activity of thymidylate synthase through the formation of the metabolite fluorodeoxyuridine monophosphate, which binds tightly, but reversibly, to the synthase enzyme.
281
It has previously been shown that the addition of vitamin B12 and folate analogues to the assay mixture may enhance [3H]TdR incorporation into D N A o1 folate and/or vitamin B12 deficient cells in a manner specific for the appropriate vitamin deficiency (Taheri et al., 1982). In general, pteroylglutamic acid (folic acid) and formyl T H F (folinic acid) correct the defect in both folate- and cobalamin-deficient cells, while 5-methyl T H F and cyanocobalamin are effective in folate and vitamin Bl2 deficiencies, respectively (Deacon et al., 1980; Van Der Weyden, 1979; Wickramasinghe, 1981; Wickramasinghe and Longland, 1974; Zittoun et al., 1978). The present study tested the ability of pteroylglutamic acid, formyl THF, 5methyl T H F and cyanocobalamin to correct abnormal d U % values caused by MTX and 5-FU. Formyl T H F and 5-methyl THF, but not cyanocobalamin and pteroylglutamic acid (results not shown), were able to correct abnormal d U % values caused by MTX. None of the correction factors tested was able to correct abnormal d U % values caused by 5-FU (results not shown). This confirms that MTX-related changes result from a folate deficiency, while 5-FU inhibits directly the thymidylate synthase activity. The dU suppression test, having been validated in-house with MTX and 5-FU, was used later to study the effect of bidisomide. Results from a recent 1-yr oral toxicity study by dietary inclusion (A. Rogister, unpublished report) suggested a potential compound-related folate and/or vitamin B~2 insufficiency in the rat. Mean morning bidisomide plasma concentrations in this study ranged from 0.2 to 14.5/~g/ml at treatment levels of 90-900 mg/kg body weight/day. These concentrations exceed largely the therapeutic range. The aim of the present study was to determine whether this potential vitamin insufficiency could affect folate-dependent DNA synthesis. The results indicate that concentrations of bidisomide in the range 1 nM to 1 mM did not affect TdR incorporation into D N A of rat bone marrow cells incubated with and without exogenous dU. This indicates that neither the folate-dependent, nor the folate-independent D N A synthesis of rat bone marrow cells is affected by bidisomide treatment in vitro. Addition of cofactors to the incubation medium did not alter the TdR incorporation (results not shown). It was concluded from these experiments that bidisomide at concentrations up to I raM, tested in vitro, does not affect directly or indirectly the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells. REFERENCES
Das K. C. and Herbert V. (1978) The lymphocyte as a marker of past nutritional status: persistence of abnormal lymphocyte deoxyuridine suppression test and chromosomes in patients with past deficiencyof folate and vitamin B12. British Journal of Haematology 38, 219-233.
282
A. ROGtSTER et al.
Das K. C. and Herbert V. (1989) In vitro DNA synthesis by megaloblastic bone marrow: effect of folates and cobalamins on thymidine incorporation and de novo thymidylate synthesis. American Journal of Hematology 31, 11-20. Das K. C., Manusselis C. and Herbert V. (1980) Simplifying lymphocyte culture and the deoxyuridine suppression test by using whole blood (0.1 ml) instead of separated lymphocytes. British Journal of Haematology 26, 72 77. Deacon R., Chanarin I., Perry J. and Lumb M. (1980) Marrow cells from patients with pernicious anaemia cannot use tetrahydrofolate normally. British Journal of Haematology 46, 523 528. Desai B. N., Cook C. S., Claypool W. D. and Garthwaite S. M. (1992) Bidisomide: Class I antiarrhythmic. Drugs of the Future 17, 374-376. Douglas K. T. (1987) The thymidylate synthesis cycle and anticancer drugs. Medicinal Research Reviews 7, 441-475. Killmann S. A. (1964) Effect of deoxyuridine on incorporation of tritiated thymidine: difference between normoblasts and megaloblasts. Acta Medica Scandinavica 175, 483-488. Matthews J. H. and Wickramasinghe S. N. (1988) The deoxyuridine suppression test performed on phyto-
haemagglutinin-stimulated peripheral blood lymphocytes fails to reflect in vivo vitamin BI2 or folate deficiency. European Journal of Haematology 40, 174-180. Taheri M. R., Wickramasinghe R. G., Jackson B. F. A. and Hoffbrand A. V. (1982) The effect of folate analogues and vitamin B12 on provision of thymidine nucleotides for DNA synthesis in megaloblastic anemia. Blood 59, 634-640. Van Der Weyden (1979) Deoxyuridine metabolism in human megaloblastic marrow cells. Scandinavian Journal of Haematology 23, 37-42. Wickramasinghe S. N. (1981) The deoxyuridine suppression test: a review of its clinical and research applications. Clinical and Laboratory Haematology 3, 1-18. Wickramasinghe S. N. and Longland J. E. (1974) Assessment of deoxyuridine suppression test in diagnosis of vitamin BI2 or folate deficiency. British Medical Journal 3, 148-150. Wickramasinghe S. N. and Matthews J. H. (1988) Deoxyuridine suppression: biochemical basis and diagnostic applications. Blood Reviews 2, 168-177. Zittoun J., Marquet J. and Zittoun R. (1978) Effect of folate and cobalamin compounds on the deoxyuridine suppression test in vitamin B12 and folate deficiency. Blood 51,
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