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A method for assessing dihydrofolate reductase inhibition in vivo
ANALYTICAL
48, 515-523
BIOCHEMISTRY
A Method
for
(1972)
Assessing
Dihydrofolate
Inhibition L. C. MISHRA, Nicrobiological X\:atiotml
in Vivo
A. S. PARMAR,
Associates, Institutes of Received
Reductase
Inc., Health.
J. A. R. MEAD
AND
and Sational Bethesda,
October
Cancer Mnrylrcntl
Institwte, X014
27, 1971
Dihydrofolate reductase, which catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid, has been found in most biological systems. The enzyme is of considerable importance because the biosynthesis of thymidylate is dependent on the regeneration of H,HF from H,F.I Also, antifolates such as methotrexate are known to inhibit this enzyme. In most studies concerning the mechanism of action of these antifolates, measurement of t’he enzyme activity has been carried out ,in vi&o using either folate (1,2) or H,F (3,4) as substrates. Since conditions existing in vitro may not entirely reflect those existing in zliwo, an attempt has been made to assess the enzyme activity in viva by administering radioactive dihydrofolate to animals pretreated with an antifolate (methotrexate) and estimating the product of the react,ion in selected organs by measuring the amount of radioactivit,y present in tetrahydrofolate. Although the met,hod does not give an estimate of the specific enzyme activity in units parallel to that obtained by the in vitro methods, it is capable of assessingt.he overall picture of enzymic activity in vivo after administration of an enzyme inhibitor. This paper describes the preparat,ion of “H-H,F and the chromat,ographic procedure used to assess H,F-reductase act,ivity and it’s inhibition by methotrexate in vivo. MATERIALS
AND
METHODS
Folic acid was obtained from Nutritional Biochemicals Company, Cleveland, Ohio, DEAE-cellulose (standard) from Carl Schleicher and Schuell Co., Keen, New Hampshire, 3’,5’-tritiated folic acid from Amersham/Searle Corp., Arlington Heights, Illinois, Cleland reagent (dithiothreitol) from Calbiochem, Gaithersburg, &Id, and methotrexate from Drug Research and Development, National Cancer Institute. The labeled folic acid contains 55% of its tritium at C-9, 35% in 3’,5’-positions, and ’ Abbreviat,ions:
F = folate;
H,F
= dihydrofolate; 515
@ 1972 hy
Academic
Press,
Inc.
H,F
= tetrahydrofolate.
516
MISHRA,
PARMAR,
AXD
MEAD
lOc/o at C-7 (5). Folic acid and methotrexatc were analyzed for purit’y by column chromatography on DEAE-cellulose and found to be 92 and 8570 pure, respect,ively. Male BDF, mice, 10 to 12 weeks old, weighing 20 to 25 gm, maintained on Purina Chow and water ud libitwz, mere used in all the experiments. A Gilson refrigerated automatic fract,ion collector equipped with an ultraviolet absorption meter, 280 nm filter, and a recorder (Recti-riter, Texas Instruments Corp.) was used for the column chromatography. An automatic gradient maker (Varigrad) was used to elute columns with linear gradients of buffers (Buchler Instruments Corp.). Since decomposition of 3H-clil~ydrofolic acid of high specific activity (170 mCi/mmolc) was found to be dependent on the levels of radioactivity (6), 3H-diliydrofolic acid of lower specific activity (8.8 mCi/ mmole) was prepared. It was found to be stable at -80°C for several mont,ha under nitrogen, and was easy to prepare with better yield and purity than that obtained from folatc of high specific activity. Tritiated folic acid, 1 mCi (1.79 mg), was mixed with 48.21 mg of folic acid in 5 ml of l.OM 2-mercaptoethanol in a 20 ml centrifuge tube and the suspension was dissolved by adding a minimum amount of 1.0 N KOH. Then 2 ml of ligroin (h.1~. 115°C) was layered over the solut’ion to protect it from air oxidation. Sodium clithionite (400 mg) was added to the solution and the reaction was carried out at 25” for 25 min with continuous, gent,le st,irring. The mixture was cooled to 0” and 0.1 M HCl was added drop by drop until the precipitation was complete. The mixture was centrifuged in a refrigeratccl centrifuge for 10 min at 2000 rpm. The yellowish white precipitate was collected and washccl twice with 2 ml of cold 0.1 $1 2-mercaptoet,hanol, s~~spcntled in 2 ml of cold distilled water, and lyophilized for 24 hr. The material was kept. in brov,n bottles under nitrogen at’mosphere at - 80”. dnnlytical procedlLre. The column chromatographic procedure to determine H?F and H,F contents in t,issues after i.p. administration of H,F in mice was essentially the same as described elsewhere (i’j. Animals were sacrificed at various time intervals after i.1~. administrat,ion of 25 mg (500 AhCi) of 3H-H,F/kg and the excised organs (2 gm) were cleaned and homogenizecl (25% w/v) in cold 0.1 M Tris (HCl) buffer, pH 7.6, containing 0.01% Cleland reagent,. The homogenate was clarified with 3 vol of ice-cold absolute ethyl alcohol and centrifuged in t,he cold at 5000 rpm for 15 min. The clear supernatant, mixed with 1 mg each of H,F and H,F as reference compounds, was slowly poured on the DEAEcellulose column. The column (1 X 10 cm) was previously washed with 100 ml of the ice-cold 0.1 M Tris buffer until a stable baseline on the UV absorption recorder was established. The elut.ion was carried out
ASSESSMENT
OF
517
H2F-REDUCThSE
first with a linear gradient, 0.1 + 0.5 Tris Jf (HCl) buffer, pH 7.6, using 100 ml of each concentration in t.he reservoirs to obtain the H,F. It was then eluted with 0.7 M Tris buffer until all the H,F appeared (7). The mean volume of the fractions n-as 5 ml in all the csperimcnts. The radioactivity present in the fractions corresponding to H,F and H,F peaks was determined by pooling the fractions in the respective peaks and counting 0.5 ml alicluots in 10 ml of Instagel-Scintillator with a Packard Tri-Carb scint.illation spectrometer model 3375. The counting efficiency for 3H was 32ycJ. The dihydrofolate rcductase ac,tioity was expressed as the radioactivity present in H,F peak as a per cent of total radioactivity present in both H,F and H.,F pcake: HAli HIF’ + H?l’ ’ RESVLTS
AND
loo >
DISCUSSION
Preparation, stability, and purity of “H-dihydrofolate. Figure 1 shows the elution pattern of F, H,F, and H,F, indicating complete separation of folate from its reduced derivatives. 3H-H2F (492,200 cpm) chromatographed similarly did not show detectable amounts of radioactivity in the H,F or folate peaks. The tot.al recovery of the radioactivity in 60 fractions was 94%, out of which 10% appeared as a peak in the first 10 fractions and 82% in the H,F, and the rest did not appear as a peak in
2 c
20
s N
40
5 5
60
z 5 F ae
80
100 IO
20
30
FRACTION
40
50
60
NO.
FIG. 1. Chromatography of F, “H-H,F, and HIF. 1 mg “H-HJ? (492.200 cpm) was mixed with approximately 1 mg F and HZ and chromntographed. Elution was first carried out with 0.0+2.0M (100 ml of each concentration) of ammonium acetate buffer (pH 7.8) and then with 2.OM buffer. Mean volume of fractions collected was 5 ml. Impurities appearing in the first 18 fractions were found in the reference HaF used and not in F and ‘H-H2F. More than 82% of the radioactivity appeared in the H,F peak.
518
MISHRA,
PARMAR,
FRACTION
AND
MEAD
NO.
FIG. 2. Separation of H,F from H,F. 1 mg each of purified HnF and HZF were mixed with liver homogenate and chromatographed. Elution was carried out first with 0.1 -0.5 M Tris buffer, pH 7.6, using 100 ml of each concentration in the reservoirs. It was then eluted with 0.7M Tris buffer until the UV-absorbing material was completely eluted.
any fraction. Figure 2 shows the separation of H,F and H,F, in which the elution was carried out according to the procedure used in all subsequent experiments using the 3H-H,F. The yield and purity with respect to 3H of the labeled H,F used in the study was always between 82-8570 and M-82%,” respectively. The radioactive impurities appeared in the first few fractions and therefore did not cause interference in the estimation of H,F and H?F. The stability of the 3H-H,F was checked OCcasionally and no evidence of decomposiGon was found during a 4 m0nt.h period. Assessment of inhibition of H2F-reductase in vivo. Although the analytical procedure used in the present study did not show any detectable decomposition of eit.her H,F or H,F (7), the possibility of biotransformation and decomposition of these compounds in viva exists. Therefore, it appeared likely that relat.ive proportions of the substrate and the product, rather than the formation of the product alone, might serve as a better criterion of dihydrofolate reductase activity and its inhibition in viva. Since maximum uptake of H,F has been shown to occur 2 hr after administration to mice (7)) the reduction of labeled H,F in liver and intestine was cletermined at the 2 hr interval. The data presented in Table 1 show that the levels of tetrahydrofolate in both ‘Note: A notification from Amersham/Searle Corp. received after complet,ion of this study indicated that the aH-folate sent to us was only 82% pure with respect to “H, which is close to the purity of the ‘H-H2F preparation found in the present study.
ASSESSMENT
Reduction
OF
519
H2F-REDUCTASE
TABLE of 3H-Uihydrofolate
1 in Liver and Intestine
dpm/gm tissuea Tissue
HZF
I-I&
Liver Intestine
1) 000
4,440
42,664 XI, 967
H*F II,F + H,F
x
100
!I7 * !I0
u Two mice were given 3H-H2F (.500 &i/kg) i.p. and sacrificed after 2 hr. Excised organs were cleaned and chrornatographed as described in the lest.
tissues were comparable and that the amount of dihydrofolate was less than 10% of the total radioactivity recovered as reduced folates. Radiochromatograms of the organs showed no evidence of any peaks which could represent cofactor forms of folate. Although the further conversion of H,F to other cofactors may occur and could be present in the H,F peak, t.his would not appreciably alter the outcome of these studies since they would necessarily hc the result of reductase action. An attempt was made to assessthe inhibition of H,F-reductase activity by a known inhibitor, methotrexate. Graded doses of methot.rexate were administered to groups of two mice each 1 hr before 3H-H,F administration; the mice were sacrificed after 2 hr. Excised liver and intestine were analyzed for amounts of radioactivity present as H,F and H,F. The total radioactivity (H,F + H,F) found in the organs did not change to any appreciable extent as a result of methot’rexate treatment. In the untreated mice the total radioactivity (H,F +H,F) in liver and intestine was 42,664 and 35,588 dpm/gm &sue, respectively. In mice treated with methotrcxate (0.5 to 15 mg/kg i.p.) the corresponding figures were 40,783 to 42,730 and 34,890 to 35,528 dpm/gm, respect,ivcly. However, methotrexate treatment caused a decrease in amount of H,F with a corresponding increase in H,F. When the per cent radioactivity in the H,F peak relat,ive to that of the t’otal radioactivity was plott’ed against the doses of methotrexate (Fig. 3)) a linear relationship between the inhibition of reduction of H,F to H,F in both tissues and the doses of methotrexate (0.5 to 15 mg/kg) was observed. Although the enzyme activity per se in both the tissues could not be directly compared, inhibition of the enzyme by the antifolate could be assessedand compared in terms of dose of the inhibitor required to cause 507$ inhibition of the enzymic activity. The ID,, for methotrexate, determined from the data in Fig. 3, was 4.5 and 10 mg/kg for intestine and liver, respectively. These values indicate that the enzymic activity in intestine was more inhibited by the drug than in liver. The enzymic activity in these two
520
MISHRA,
PARMAR,
Ah-D
MEAD
JO--
20--
IO--
0’
I 25
/ 50 METHOTREXATE
I 75
I 100
I 125
I 150
I 175
(MG/KG)
FIG. 3. Effect of methotrexate on HzF-reductase activity in mouse liver (0) and intestine (0) in vivo. Two mice were given “H-HZF, 25 mg (500 pCi)/kg i.p., 1 hr after methotrexate trcatmcnt and sacrificed after 2 hr.
organs was also studied at various time intervals after pret,reatment with methotrexate. The data in Fig. 4 show that, indeed, the enzyme was more inhibited in inte&ine than liver and that the difference continued to exist for a period of more than 48 hr, during which enzyme act,ivity was increasing. These experiments revealed an interesting biphasic recovery of the enzyme activity in both the gut, and the liver; a rapid recovery up to 45 and 72% wit~hin 6 hr followed by a slow recovery up to 92 and 9870, respectively within 72 hr. These experiments confirmed the differences in the inhihit.ion of the enzyme in the two organs obtained in the previous experiment’. Zakrzewski and Himberg (6) recently described the preparation and purification of 3H-H,F of high specific activity and its use in estimating H,F-reductase activity. They encountered considerable decomposition of their radioactive material and suggested that t’he decomposition may be due in part to t,he high specific activity of the preparation. The method also involved lyophilization of large volumes of ammonium bicarbonate buffer, which may also result in some clecomposit,ion. Their procedure for estimat’ing H,F-reductase was primarily based on estimation
521
HOURS OF PRETREATMENT WITH MTX
FIG. 4. Recovery of H?F-reductase in mouse liver (0) and intestine (0) in uivo after methotrexate treatment. Two mice were given “H-H,F, 25 m g (500 &N/kg, at various time intcrrals after mcthotresate (15 mg/kg i.p.) treatment and sacrificed after 2 hr.
of unchanged tritiated H,F. It is likely that, under some experimental condit,ions, the decrease in 3H-H,F might be associated with nonreductase transformations. For this reason, estimation of both H,F and H,F was carried out in the present study. The inhibition of the enzyme in liver observed in this study was comparable t.o that presented elsewhere (7) in which large doses of H,F (400 mg/kg) were injected so that sufficient quantities of H,F could be isolated from 1 to 2 gm of liver for estimation and characterization by UV absorption spectrum. h lesser dose of H,F (25 mg/kg) did not, shoTy any significant difference in the inhibition and recovery of the enzyme after methotrexate t.reatment than &at observed previously (7) with the larger dose (400 mg/kg) . It seemslikely that assessmentof inhibition of the enzyme in Z/~LJO is not greatly influenced by displacement of methotrexate from the enzyme (8) by large amounts of H,F. The data. presented here suggest that, the procedure may be applied to estimating the effect of antifolates on H,F-reductase in zlivo and may help in elucidating their mechanism of action. For example, the procedure
522
MISHRA,
PARMAR,
AND
1fEAD
might provide an opportunity to correlate the inhibition of nucleic acid and protein biosynthesis in viva with the in viwo inhibition of H,F-reductase activity after administration of an antifolate. Poor correlation between the inhibition of H,F-reductase assessed by the in vitro assay and the uptake of deoxyuridine into DNA after methotrexate administration has been reported (9-11). It is possible that because of compartmentation in cells the drug may not reach all the enzyme throughout the cell and, thus, some enzyme activity may actually escape inhibition. This could not be assessedby the i?z vitro assay method since it requires disintegration of the cells to isolate the enzyme and would allow any free drug to inhibit the previously uninhibited enzyme. SUMMARY
A method for studying inhibition of dihydrofolate reductase activity in z&o has been described. &lice were given 500 &i (25 mg) 3H-HzF/kg intraperitoneally, the tetrahydrofolate (H,F) formed in liver and gut were separated by DEAE-cellulose column chromatography of tissue homogenates, and the radioact,ivity in peaks corresponding to H,F and H,F was determined. A linear relationship was observed in the inhibition of H,F-reductase by methotrexate in both liver and intestine when the mice were treated wit.h methotrexate 1 hr before H?F injection and the tissues were assayed 2 hr later. The recovery of enzyme activity from inhibition by met,hotrexate in gut and liver was biphasic: a rapid recovery (45% in gut, and 72% in liver) within 6 hr was followed by a slower recovery (92% in gut and 98% in liver) in 2 to 3 days. The data presented indicate greater inhibition and slower recovery of H,F-reductase in intestine than in liver and illustrate t,he feasibility of assessing H,F rcductase activity in viva. ACKKOWLEDGMENTS
The authorsare most grateful to Dr. R. D. Costlowfor his valuable suggestions. The study was in part supportedby contract PH 43-68-1283 Chemotherapy, NaGonalCancerInstitute, NIH, Bethesda,Maryland 20014. REFERENCES Biochemistry 5, 3549 WERKHEISER, W. C., ZAKRZEWSKI, Therap. 137, 162 (1962). OSBORN, M. J., FREEMAN, M., AND Med. 97, 429 (1958). OSBORN, M. J., AND HUENNEKENS, ZAKRZEWSKI, S. F., EVANS, E. A.. (1970). ZAKRZEWSKI, S. F., AND HIMBERG,
1. ROBERTS, 2.
3. 4.
5. 6.
D.,
(1966). S. F., AND
F. M.,
HLJEIYNEKENS,
F. M., AND
C. A., J. Pharmacol.
XICHOL,
J. Biol.
PHILLIPS,
J. J., Anal.
hoc.
Sot.
ExpZ.
Chem. 233, 969 (19%). R. F., Anal. Biochem. 36,
Biochem.
40, 336
(1971).
Expl. Biol.
197
ASSESSMENT
2871,
9.
10. 11.
H?F-REDUCTASE
523
L. C., PARM.~R, A. S., AND MEAD, J. A. R., Biochem. Phnrnmcol. 20, (1971). JOHNS, D. G., HOLLINGSWORTH, J. W.. CASHMORE, A. R., PLENDERLEITH, I. H., AND BERTINO, J. R., J. Clin. Invest. 43, 621, (1964). ISHIHARA, S., AND CONDIT, P. T., Proc. Amer. Assoc. Cnmer Res. 12, 36 (1971). ROBERTS, D., AND WODINSKY, I., Cancer Iles. 28, 1955 (1968). MARGOLIS, S., PHILIPS, F. S.. AND STERPI’BERG, S. S., Cawxr Res. 31, 2037 (1971)
7. MISHRA, 8.
OF
48, 515-523
BIOCHEMISTRY
A Method
for
(1972)
Assessing
Dihydrofolate
Inhibition L. C. MISHRA, Nicrobiological X\:atiotml
in Vivo
A. S. PARMAR,
Associates, Institutes of Received
Reductase
Inc., Health.
J. A. R. MEAD
AND
and Sational Bethesda,
October
Cancer Mnrylrcntl
Institwte, X014
27, 1971
Dihydrofolate reductase, which catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid, has been found in most biological systems. The enzyme is of considerable importance because the biosynthesis of thymidylate is dependent on the regeneration of H,HF from H,F.I Also, antifolates such as methotrexate are known to inhibit this enzyme. In most studies concerning the mechanism of action of these antifolates, measurement of t’he enzyme activity has been carried out ,in vi&o using either folate (1,2) or H,F (3,4) as substrates. Since conditions existing in vitro may not entirely reflect those existing in zliwo, an attempt has been made to assess the enzyme activity in viva by administering radioactive dihydrofolate to animals pretreated with an antifolate (methotrexate) and estimating the product of the react,ion in selected organs by measuring the amount of radioactivit,y present in tetrahydrofolate. Although the met,hod does not give an estimate of the specific enzyme activity in units parallel to that obtained by the in vitro methods, it is capable of assessingt.he overall picture of enzymic activity in vivo after administration of an enzyme inhibitor. This paper describes the preparat,ion of “H-H,F and the chromat,ographic procedure used to assess H,F-reductase act,ivity and it’s inhibition by methotrexate in vivo. MATERIALS
AND
METHODS
Folic acid was obtained from Nutritional Biochemicals Company, Cleveland, Ohio, DEAE-cellulose (standard) from Carl Schleicher and Schuell Co., Keen, New Hampshire, 3’,5’-tritiated folic acid from Amersham/Searle Corp., Arlington Heights, Illinois, Cleland reagent (dithiothreitol) from Calbiochem, Gaithersburg, &Id, and methotrexate from Drug Research and Development, National Cancer Institute. The labeled folic acid contains 55% of its tritium at C-9, 35% in 3’,5’-positions, and ’ Abbreviat,ions:
F = folate;
H,F
= dihydrofolate; 515
@ 1972 hy
Academic
Press,
Inc.
H,F
= tetrahydrofolate.
516
MISHRA,
PARMAR,
AXD
MEAD
lOc/o at C-7 (5). Folic acid and methotrexatc were analyzed for purit’y by column chromatography on DEAE-cellulose and found to be 92 and 8570 pure, respect,ively. Male BDF, mice, 10 to 12 weeks old, weighing 20 to 25 gm, maintained on Purina Chow and water ud libitwz, mere used in all the experiments. A Gilson refrigerated automatic fract,ion collector equipped with an ultraviolet absorption meter, 280 nm filter, and a recorder (Recti-riter, Texas Instruments Corp.) was used for the column chromatography. An automatic gradient maker (Varigrad) was used to elute columns with linear gradients of buffers (Buchler Instruments Corp.). Since decomposition of 3H-clil~ydrofolic acid of high specific activity (170 mCi/mmolc) was found to be dependent on the levels of radioactivity (6), 3H-diliydrofolic acid of lower specific activity (8.8 mCi/ mmole) was prepared. It was found to be stable at -80°C for several mont,ha under nitrogen, and was easy to prepare with better yield and purity than that obtained from folatc of high specific activity. Tritiated folic acid, 1 mCi (1.79 mg), was mixed with 48.21 mg of folic acid in 5 ml of l.OM 2-mercaptoethanol in a 20 ml centrifuge tube and the suspension was dissolved by adding a minimum amount of 1.0 N KOH. Then 2 ml of ligroin (h.1~. 115°C) was layered over the solut’ion to protect it from air oxidation. Sodium clithionite (400 mg) was added to the solution and the reaction was carried out at 25” for 25 min with continuous, gent,le st,irring. The mixture was cooled to 0” and 0.1 M HCl was added drop by drop until the precipitation was complete. The mixture was centrifuged in a refrigeratccl centrifuge for 10 min at 2000 rpm. The yellowish white precipitate was collected and washccl twice with 2 ml of cold 0.1 $1 2-mercaptoet,hanol, s~~spcntled in 2 ml of cold distilled water, and lyophilized for 24 hr. The material was kept. in brov,n bottles under nitrogen at’mosphere at - 80”. dnnlytical procedlLre. The column chromatographic procedure to determine H?F and H,F contents in t,issues after i.p. administration of H,F in mice was essentially the same as described elsewhere (i’j. Animals were sacrificed at various time intervals after i.1~. administrat,ion of 25 mg (500 AhCi) of 3H-H,F/kg and the excised organs (2 gm) were cleaned and homogenizecl (25% w/v) in cold 0.1 M Tris (HCl) buffer, pH 7.6, containing 0.01% Cleland reagent,. The homogenate was clarified with 3 vol of ice-cold absolute ethyl alcohol and centrifuged in t,he cold at 5000 rpm for 15 min. The clear supernatant, mixed with 1 mg each of H,F and H,F as reference compounds, was slowly poured on the DEAEcellulose column. The column (1 X 10 cm) was previously washed with 100 ml of the ice-cold 0.1 M Tris buffer until a stable baseline on the UV absorption recorder was established. The elut.ion was carried out
ASSESSMENT
OF
517
H2F-REDUCThSE
first with a linear gradient, 0.1 + 0.5 Tris Jf (HCl) buffer, pH 7.6, using 100 ml of each concentration in t.he reservoirs to obtain the H,F. It was then eluted with 0.7 M Tris buffer until all the H,F appeared (7). The mean volume of the fractions n-as 5 ml in all the csperimcnts. The radioactivity present in the fractions corresponding to H,F and H,F peaks was determined by pooling the fractions in the respective peaks and counting 0.5 ml alicluots in 10 ml of Instagel-Scintillator with a Packard Tri-Carb scint.illation spectrometer model 3375. The counting efficiency for 3H was 32ycJ. The dihydrofolate rcductase ac,tioity was expressed as the radioactivity present in H,F peak as a per cent of total radioactivity present in both H,F and H.,F pcake: HAli HIF’ + H?l’ ’ RESVLTS
AND
loo >
DISCUSSION
Preparation, stability, and purity of “H-dihydrofolate. Figure 1 shows the elution pattern of F, H,F, and H,F, indicating complete separation of folate from its reduced derivatives. 3H-H2F (492,200 cpm) chromatographed similarly did not show detectable amounts of radioactivity in the H,F or folate peaks. The tot.al recovery of the radioactivity in 60 fractions was 94%, out of which 10% appeared as a peak in the first 10 fractions and 82% in the H,F, and the rest did not appear as a peak in
2 c
20
s N
40
5 5
60
z 5 F ae
80
100 IO
20
30
FRACTION
40
50
60
NO.
FIG. 1. Chromatography of F, “H-H,F, and HIF. 1 mg “H-HJ? (492.200 cpm) was mixed with approximately 1 mg F and HZ and chromntographed. Elution was first carried out with 0.0+2.0M (100 ml of each concentration) of ammonium acetate buffer (pH 7.8) and then with 2.OM buffer. Mean volume of fractions collected was 5 ml. Impurities appearing in the first 18 fractions were found in the reference HaF used and not in F and ‘H-H2F. More than 82% of the radioactivity appeared in the H,F peak.
518
MISHRA,
PARMAR,
FRACTION
AND
MEAD
NO.
FIG. 2. Separation of H,F from H,F. 1 mg each of purified HnF and HZF were mixed with liver homogenate and chromatographed. Elution was carried out first with 0.1 -0.5 M Tris buffer, pH 7.6, using 100 ml of each concentration in the reservoirs. It was then eluted with 0.7M Tris buffer until the UV-absorbing material was completely eluted.
any fraction. Figure 2 shows the separation of H,F and H,F, in which the elution was carried out according to the procedure used in all subsequent experiments using the 3H-H,F. The yield and purity with respect to 3H of the labeled H,F used in the study was always between 82-8570 and M-82%,” respectively. The radioactive impurities appeared in the first few fractions and therefore did not cause interference in the estimation of H,F and H?F. The stability of the 3H-H,F was checked OCcasionally and no evidence of decomposiGon was found during a 4 m0nt.h period. Assessment of inhibition of H2F-reductase in vivo. Although the analytical procedure used in the present study did not show any detectable decomposition of eit.her H,F or H,F (7), the possibility of biotransformation and decomposition of these compounds in viva exists. Therefore, it appeared likely that relat.ive proportions of the substrate and the product, rather than the formation of the product alone, might serve as a better criterion of dihydrofolate reductase activity and its inhibition in viva. Since maximum uptake of H,F has been shown to occur 2 hr after administration to mice (7)) the reduction of labeled H,F in liver and intestine was cletermined at the 2 hr interval. The data presented in Table 1 show that the levels of tetrahydrofolate in both ‘Note: A notification from Amersham/Searle Corp. received after complet,ion of this study indicated that the aH-folate sent to us was only 82% pure with respect to “H, which is close to the purity of the ‘H-H2F preparation found in the present study.
ASSESSMENT
Reduction
OF
519
H2F-REDUCTASE
TABLE of 3H-Uihydrofolate
1 in Liver and Intestine
dpm/gm tissuea Tissue
HZF
I-I&
Liver Intestine
1) 000
4,440
42,664 XI, 967
H*F II,F + H,F
x
100
!I7 * !I0
u Two mice were given 3H-H2F (.500 &i/kg) i.p. and sacrificed after 2 hr. Excised organs were cleaned and chrornatographed as described in the lest.
tissues were comparable and that the amount of dihydrofolate was less than 10% of the total radioactivity recovered as reduced folates. Radiochromatograms of the organs showed no evidence of any peaks which could represent cofactor forms of folate. Although the further conversion of H,F to other cofactors may occur and could be present in the H,F peak, t.his would not appreciably alter the outcome of these studies since they would necessarily hc the result of reductase action. An attempt was made to assessthe inhibition of H,F-reductase activity by a known inhibitor, methotrexate. Graded doses of methot.rexate were administered to groups of two mice each 1 hr before 3H-H,F administration; the mice were sacrificed after 2 hr. Excised liver and intestine were analyzed for amounts of radioactivity present as H,F and H,F. The total radioactivity (H,F + H,F) found in the organs did not change to any appreciable extent as a result of methot’rexate treatment. In the untreated mice the total radioactivity (H,F +H,F) in liver and intestine was 42,664 and 35,588 dpm/gm &sue, respectively. In mice treated with methotrcxate (0.5 to 15 mg/kg i.p.) the corresponding figures were 40,783 to 42,730 and 34,890 to 35,528 dpm/gm, respect,ivcly. However, methotrexate treatment caused a decrease in amount of H,F with a corresponding increase in H,F. When the per cent radioactivity in the H,F peak relat,ive to that of the t’otal radioactivity was plott’ed against the doses of methotrexate (Fig. 3)) a linear relationship between the inhibition of reduction of H,F to H,F in both tissues and the doses of methotrexate (0.5 to 15 mg/kg) was observed. Although the enzyme activity per se in both the tissues could not be directly compared, inhibition of the enzyme by the antifolate could be assessedand compared in terms of dose of the inhibitor required to cause 507$ inhibition of the enzymic activity. The ID,, for methotrexate, determined from the data in Fig. 3, was 4.5 and 10 mg/kg for intestine and liver, respectively. These values indicate that the enzymic activity in intestine was more inhibited by the drug than in liver. The enzymic activity in these two
520
MISHRA,
PARMAR,
Ah-D
MEAD
JO--
20--
IO--
0’
I 25
/ 50 METHOTREXATE
I 75
I 100
I 125
I 150
I 175
(MG/KG)
FIG. 3. Effect of methotrexate on HzF-reductase activity in mouse liver (0) and intestine (0) in vivo. Two mice were given “H-HZF, 25 mg (500 pCi)/kg i.p., 1 hr after methotrexate trcatmcnt and sacrificed after 2 hr.
organs was also studied at various time intervals after pret,reatment with methotrexate. The data in Fig. 4 show that, indeed, the enzyme was more inhibited in inte&ine than liver and that the difference continued to exist for a period of more than 48 hr, during which enzyme act,ivity was increasing. These experiments revealed an interesting biphasic recovery of the enzyme activity in both the gut, and the liver; a rapid recovery up to 45 and 72% wit~hin 6 hr followed by a slow recovery up to 92 and 9870, respectively within 72 hr. These experiments confirmed the differences in the inhihit.ion of the enzyme in the two organs obtained in the previous experiment’. Zakrzewski and Himberg (6) recently described the preparation and purification of 3H-H,F of high specific activity and its use in estimating H,F-reductase activity. They encountered considerable decomposition of their radioactive material and suggested that t’he decomposition may be due in part to t,he high specific activity of the preparation. The method also involved lyophilization of large volumes of ammonium bicarbonate buffer, which may also result in some clecomposit,ion. Their procedure for estimat’ing H,F-reductase was primarily based on estimation
521
HOURS OF PRETREATMENT WITH MTX
FIG. 4. Recovery of H?F-reductase in mouse liver (0) and intestine (0) in uivo after methotrexate treatment. Two mice were given “H-H,F, 25 m g (500 &N/kg, at various time intcrrals after mcthotresate (15 mg/kg i.p.) treatment and sacrificed after 2 hr.
of unchanged tritiated H,F. It is likely that, under some experimental condit,ions, the decrease in 3H-H,F might be associated with nonreductase transformations. For this reason, estimation of both H,F and H,F was carried out in the present study. The inhibition of the enzyme in liver observed in this study was comparable t.o that presented elsewhere (7) in which large doses of H,F (400 mg/kg) were injected so that sufficient quantities of H,F could be isolated from 1 to 2 gm of liver for estimation and characterization by UV absorption spectrum. h lesser dose of H,F (25 mg/kg) did not, shoTy any significant difference in the inhibition and recovery of the enzyme after methotrexate t.reatment than &at observed previously (7) with the larger dose (400 mg/kg) . It seemslikely that assessmentof inhibition of the enzyme in Z/~LJO is not greatly influenced by displacement of methotrexate from the enzyme (8) by large amounts of H,F. The data. presented here suggest that, the procedure may be applied to estimating the effect of antifolates on H,F-reductase in zlivo and may help in elucidating their mechanism of action. For example, the procedure
522
MISHRA,
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AND
1fEAD
might provide an opportunity to correlate the inhibition of nucleic acid and protein biosynthesis in viva with the in viwo inhibition of H,F-reductase activity after administration of an antifolate. Poor correlation between the inhibition of H,F-reductase assessed by the in vitro assay and the uptake of deoxyuridine into DNA after methotrexate administration has been reported (9-11). It is possible that because of compartmentation in cells the drug may not reach all the enzyme throughout the cell and, thus, some enzyme activity may actually escape inhibition. This could not be assessedby the i?z vitro assay method since it requires disintegration of the cells to isolate the enzyme and would allow any free drug to inhibit the previously uninhibited enzyme. SUMMARY
A method for studying inhibition of dihydrofolate reductase activity in z&o has been described. &lice were given 500 &i (25 mg) 3H-HzF/kg intraperitoneally, the tetrahydrofolate (H,F) formed in liver and gut were separated by DEAE-cellulose column chromatography of tissue homogenates, and the radioact,ivity in peaks corresponding to H,F and H,F was determined. A linear relationship was observed in the inhibition of H,F-reductase by methotrexate in both liver and intestine when the mice were treated wit.h methotrexate 1 hr before H?F injection and the tissues were assayed 2 hr later. The recovery of enzyme activity from inhibition by met,hotrexate in gut and liver was biphasic: a rapid recovery (45% in gut, and 72% in liver) within 6 hr was followed by a slower recovery (92% in gut and 98% in liver) in 2 to 3 days. The data presented indicate greater inhibition and slower recovery of H,F-reductase in intestine than in liver and illustrate t,he feasibility of assessing H,F rcductase activity in viva. ACKKOWLEDGMENTS
The authorsare most grateful to Dr. R. D. Costlowfor his valuable suggestions. The study was in part supportedby contract PH 43-68-1283 Chemotherapy, NaGonalCancerInstitute, NIH, Bethesda,Maryland 20014. REFERENCES Biochemistry 5, 3549 WERKHEISER, W. C., ZAKRZEWSKI, Therap. 137, 162 (1962). OSBORN, M. J., FREEMAN, M., AND Med. 97, 429 (1958). OSBORN, M. J., AND HUENNEKENS, ZAKRZEWSKI, S. F., EVANS, E. A.. (1970). ZAKRZEWSKI, S. F., AND HIMBERG,
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L. C., PARM.~R, A. S., AND MEAD, J. A. R., Biochem. Phnrnmcol. 20, (1971). JOHNS, D. G., HOLLINGSWORTH, J. W.. CASHMORE, A. R., PLENDERLEITH, I. H., AND BERTINO, J. R., J. Clin. Invest. 43, 621, (1964). ISHIHARA, S., AND CONDIT, P. T., Proc. Amer. Assoc. Cnmer Res. 12, 36 (1971). ROBERTS, D., AND WODINSKY, I., Cancer Iles. 28, 1955 (1968). MARGOLIS, S., PHILIPS, F. S.. AND STERPI’BERG, S. S., Cawxr Res. 31, 2037 (1971)
7. MISHRA, 8.
OF