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Comparative effects of ftorafur and 5-fluorouracil on DNA synthesis in rat small intestine
Life Scisacas Vol. 17, pp . Printed in the U.S .A .
Pergamon Press
1363-1368
COMPARATIVE EFFECTS OF FTORAFUR AND 5-FLUOROURACIL ON DNA SYNTHESIS IN RAT SMALL INTESTINE Arthur M . Cohen, Ph .D . Department of Biopharmaceutical Sciences, College of Pharmacy University of Utah ; Salt Lake City, Utah 84112 (Received in final form Anguet 4, 1975)
~mma~y The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the in vivo incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied . 5fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection . This degree of inhibition was sustained for at least 12 hours . Deoxyuridine inwrporation was inhibited by 30 to 65`X, during the initial six hours after the injection of ftorafur . By 12 hours the rate of incorporation had returned m 66916 of the control value . Neither drug inhibited thymidine incorporation into DNA . A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine . The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed . Ftorafur (Ft), 1-(tetrahydrofuran-2-yl)-5-fluorouracil, was synthesized in the Soviet Union by Hiller, et g~ . , as part of a program designed to study the antitumor effect of nucleosides containing modified sugar and heterocyclic moieties (1) . Clinical studies have indicated that Ft can induce regressions of both primary tumors and metastases in patients with carcinoma of the breast, colon, or rectum (2) . Moreover, these objective responses are apparently associated with a lower incidence of gastrointestinal and bone marrow toxicity than is observed after treatment with 5-fluorouracil (5-FU) . Recent studies in mice have also indicated that Ft is less toxic than 5-FU to hematopoietic stem cells (3) . In oraler to better understand the differences in the toxicological properties of Ft and 5-FU, a study was made of the effects of equimolar doses of the two drugs on in vim, DNA synthesis in the small intestine of the rat . In addition, the in vivo uptake and metabolic disposition of the two drugs in the intestine was studied . The aesults of these investigations indicate that the difference in cytotoxic activity between Ft and 5-FU may be related to the more complete conversion of the latter drug into fluorouracil-containing nucleotides by normal proliferating host tissues .
1363
1364
Sffact of Ftorafur aad 5-Fluorouracil
Vol. 17, No . 9
Methods Male, Sprague-Dawley rats were given intravenous injections of Ft or 5-FU dissolved in 0 .2 M sodium bicarbonate/carbonate, pH 9 .5 . Control anlmala receivéd an equal volume of the buffer alone . At various intervals thereafter they received an intravenous injection of either (6-3H) deoxyuridine (76 ~ Cükq, specific activity, 21 ~ Ci/G,i mole) or (methyl- 3H) thymidine (80 ~ Ci/kq ; specific activity, 40 u Ci/u mole) and were killed 20 or 10 minutes later, respectively . The small intestine was removed and placed in ice-cold 0 .996 NaCI . The intestines were then slit open and agitated to remove fecal material . DNA was extracted from homogenates of the cleaned intestines by the procedure of Munro and Fleck (4) . The DNA content of the extracts were determined colorimetrically according to the method of Schneider (5) with deoxyadenosine serving as a standard . Radioactivity present in the extracts was determined by liquid scintillation spectrometry . Counting efficiency was estimated by internal standardization . (2- 14 C) ftorafur was prepared by Dr . Leroy B . Townsend, University of Utah (6) . (2- 14 C) 5-fluorouracil was purchased from Caltomic . The radiochemical purity of both compounds was found to be > 9796 following separation by the thin layer chromatography procedure deacrlbed below . Acid soluble metabolites were extracted from the small intestine at 0 .5 and 4 hours following the intravenous injection of (2- 14 C) Ft (50 ~ Ci/kq; specific activity, 50 ~ C!/100 mg) or (2- 14C) 5-FU (48 ~ Ci/kg; specific activity, 48 ~ Cî/65 mq) . The intestines were homogenized in three volumes of ice-cold water and an equal volume of wld 0 .4N perchloric acid was added . The acid insoluble material was removed by centrifugation and the supernatant was saved . The sediment was washed twice with 0 .2N perchloric acid and the washings were added to the original supernatant. The combined supernatant was then adjusted to pH 7 with potassium hydroxide . After allowing the neutralized extract to remain in the cold for several hours, the insoluble potassium perchlorate was removed by centrifugation . The extract was concentrated by freeze-drying and the residue was dissolved in a small volume of water . The metabolües present in the neu~alized extract were separated on silica gel chromatography plates containing a fluorescent indicator (Chromar 7 GF ; Mallinckrodt) . The location of the metabolites was facilitated by the use of non labeled standards which were spotted on top of the radioactive samples . These standards were then located under U .V . light after the chromatogram was developed . In all cases a duplicate radioactive sample was spotted ahead of the solvent front to allow for the determination of recovery of radioactivity from the chromatogram . The solvent system employed consisted of ethyl acetate: methanol : conc . NH40H (75 :25:1, V/V/V) . The Rf of the metabolites were as follows : Ft = 0 .74; 5-FU 0 .58; 5-fluoro-2'-deoxyuridine (FUdR) = 0 .47; 5-fluorouridine (FUR) = 0 .25 . Nucleotides (5-fluoro-2'-deoxyuridine-5'-monophoaphate and 5-fluorouridine-5'- ' monophoaphate) remained at the origin . Following development, each spot was scraped off, eluted with water and counted by liquid scintillation spectrometry . Counting efficiency was estimated by internal standardization . Results and Disc ussion Figure 1 shows the effect of equimolar doses of Ft (100 mg/'kq) and 5-FU (65 mq/kq) on the in vive incorporation of (6-3H) deoxyuridine into the DNA of the small intestine . Ft produced a 30-5096 decrease, in the rate of incorporation at one-two hours after intravenous injection. In the subsequent two hours the
Effect of Ftorafur and 5-Fluorouracil
Vol . 17, No . 9
1365
ts~
ta~
2
HOURS AFTER LV DOSE
FIG . 1 Effect of ftorafur and 5-fluorouracil on the incorporation of (6-3H) deoxyuridine into intestinal DNA. Each symbol is the mean specific activity of the DNA extinct and must be multiplied by 100 to obtain the actual DPM/(1 mole deoxyriboae . The vertical bars show the S .E, of the wean . Numbers in parenthesis denote the number of animals per treatment group . Asterisks indicate that the value for the treatment group differed significantly (p ~ 0 .05) from the control group . O, control; / ftorafur (100 mg/kq) ; ~, ftorafur (300 mg/kq) ; rate of incorporation decreased tD about 3596 of the control value . Thereafter, deoxyuridine incorporation began to increase ao that by 12 hours the rate was 66% of the control value . In contrast, an equimolar dose of 5-FU produced a greater than 90% inhibition in the rate of deoxyuridinè incorporation within one hour . Moreover, this degree of inhibition was sustained for at least 12 hours . Fig . 1 also shows that the administration of Ft at a dose of 300 mg/kg produced effects similar to those observed with 5-FU . However, even at this high dose level, maximum inhibition of deoxyuridine incorporation was not attained until two hours after injection of the drug . 5-FU is believed to exert its antitumor and toxic effects after being converted ill, vi to 5-fluoro-2'-deoxyuridine-5'-monophosphate which is a potent inhibitor of thymidylate synthetase (7,8) . Since Fti is an analog of 5-FU, it we~ of irnerest to determine if the thymidylate synthetase reaction was specifically inhibited by this new drug . Accordingly, the effect of Ft {300 mg/kg) on the ~l.yjYS incorporation of (methyl- 3 H) thymidine into intestinal DNA was measured at two and
1366
8ffect of !`torafur sad S-L~luorouracil
Vol . 17, No . 9
four hours after injection of the drug . It will be rocalled from Fiq. 1 that deoxyuridine incorporation was maximally inhibited by this dose of Ft at both of these time periods . The data in Table 1 show that in contrast to its effects on deoxyuridine incorporation at two hours , Ft did not significantly inhibit thymidine inwrTABLE 1 Effect of Ftorafur and 5-FU on the Incorporation of Thymidine into DNA of Rat Small Intestine l2PM/k M~e Deoxytibos~a
~I$. Control Ft(300 mg/kq) S-FU(65 mq/kq)
1868 _+ 398 (4) 1768 _+ 290 (4) 2193 + 259 (3)
1853 ± 134 (3) 3256 ± 323 (5) 2983 + 670 (4)
aMean ± S .E . The numbers in parenthesis rofer to the number of animals per treatment group . potation into DNA . At four hours a stimulation of thymidine incorporation was observed . This most likely reflected a decrease in the endogenous thymidine nucleotide pools which would oth®rwise be available to dilute the radioacttve thymidine infected into the rats . Table 1 also shows that similar rosults were obtained after infection of 5-FU and thus indicates that both drugs sham at least one common biochemical site of action . Since the observed differoncea in the effects of equimolar doses of Ft and 5-FU on deoxyuridine incorporation into DNA (Fig . 1) might have been rolated to differonces in the metabolic disposition of the two drugs, a study was made of the incorporation of radioactivity labeled Ft and 5-FU into the acid-soluble fraction of the small intestine . The data in Table 2 show that the fatal concentration of each drug and its metabolites were nearly equivalent at 30 minutes after the infection of equimolar doses of the two drugs . Ap~oximately 6596 of the total amount present after the infection of Ft was accounted for by the unmetabolized parent compound . Leas than 2096 of the drug had been converted to fluorouracil-containing nucleosides and nucleotides . In contrast, less than 259K of the total drug equivalents present in the extracts from rats given 5-FU was due m the unmetabolized drug . Furthermoro, fluorouracil-containing nucleotides accounted for nearly S09I6 of the total radioactivity present. In absolute terms , between throe and four times as much 5-FU was converted to nucleotides in the small intestine within 30 minutes after infection of equimolar doses of the two drugs . At four hours after the infection of Ft little change was observed in either the total concentration of drug equivalents or the distribution of metabolites . In contrast, the total concentration of drug equivalents from 5-FU was about half of that found at 30 minutes . However, fluorouracil-containing nucleotides still accounted for appraximaiely 5096 of the total radioactivity present . Theroforo, in absolute terms, the concentration of these nucleotides was about twice as great following 5-FU than after the infection of an equivalent amount of Ft . The prosent studies indicate that the reported lower toxicity of Ft as compared to 5-FU may be related to a relatively slow rate of conversion of the former drug into fluorouracil-containing nucleotides by normal proliferating hoot
Vol.
17,
No . 9
Effect of Ftorafur an8 5-Fluorouracil
1367
TABLE 2 Thin Layer Chromatography of Acid-Soluble Metabolites in Small Intestines METABOLITE CONCENTRATION (n mole/gm) Q" 5 hr
5-FU 213
Ft 195
4 t~
5-FU 97
TOTAL
Ft 230
Ft FU FUdR FUR Nucleotides
150 18 5 2 30
50 17 11 103
157 8 2 2 25
22 5 7 51
Recovery(%)
89
85
100
89
Pairs of rats received an intravenous injection of either (2- 14 C) ftomfur (50 u Ci/kg ; specific activity 50 ~ Ci/100 mg) or (2-14C) 5-fluorouridine (48 ~ Ci/kg; specific activity 48 ~t Ci/65 mg) . The rats were killed at 0 .5 or 4 hours thereafter and the acidsoluble metabolites were extracted and separated on silica gel plates as described in the text . Each value is the mean of a single pair of rata . tissues . 5-FU is believed to inhibit DNA synthesis and exert its cytotoxic effects only after it has been converted to 5-fluoro-2'-deaucyuridine-5'-monophoaphate . This nucleotide is a potent inhibitor of the enzyme, thymidglate aynthetase (7,8) . The more rapid and marked inhibition of deoxyuridine incorporation into the DNA of the small intestine after the administration of 5-FU as compared to Ft (Fig . 1) is aonsistant with the more complete conversion of the former drug to the nucleotide level ~ vivo (Table 2 ) . ~knowled2ement This work was supported by United States Public Health Service Grant CA 13238 . ~efe~ences 1 . S . A . H111er, R . A. Spuk , and M . Y. Lidak , Dokl . Adad . Nauk . (USSR), 176, 332-335 (1967) . 2 . N . G . Blokhina, E . K . Vozny and A . M . Garin, Cancer, 30, 390-392 (1972) . 3 . I. Hrsak, and S . Pavicic, Biomedicine , 21, 164-67 (1974) . 4 . H . N . Munro, and A . Fleck, Meth . Biochem . Anal ., 14, 112-176 (1966) . 5 . W. C . Schneider, Meth . Enzvmol., 3, 680-684 (1957) . 6 . R. A . Earl and L . B . Townsend, T. Heterocvcl . Chem ., 9, 1141-1143 (1972) . 7 . I. U . Hartmann, and C . Heidelberger, T . Biol . Chem ., 23fi, 2006-3013 (1961) . 8 . P . Reyes, and C . Heidelberger, Mol. Pharmacol., 1, 14-30 (1965) .
Pergamon Press
1363-1368
COMPARATIVE EFFECTS OF FTORAFUR AND 5-FLUOROURACIL ON DNA SYNTHESIS IN RAT SMALL INTESTINE Arthur M . Cohen, Ph .D . Department of Biopharmaceutical Sciences, College of Pharmacy University of Utah ; Salt Lake City, Utah 84112 (Received in final form Anguet 4, 1975)
~mma~y The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the in vivo incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied . 5fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection . This degree of inhibition was sustained for at least 12 hours . Deoxyuridine inwrporation was inhibited by 30 to 65`X, during the initial six hours after the injection of ftorafur . By 12 hours the rate of incorporation had returned m 66916 of the control value . Neither drug inhibited thymidine incorporation into DNA . A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine . The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed . Ftorafur (Ft), 1-(tetrahydrofuran-2-yl)-5-fluorouracil, was synthesized in the Soviet Union by Hiller, et g~ . , as part of a program designed to study the antitumor effect of nucleosides containing modified sugar and heterocyclic moieties (1) . Clinical studies have indicated that Ft can induce regressions of both primary tumors and metastases in patients with carcinoma of the breast, colon, or rectum (2) . Moreover, these objective responses are apparently associated with a lower incidence of gastrointestinal and bone marrow toxicity than is observed after treatment with 5-fluorouracil (5-FU) . Recent studies in mice have also indicated that Ft is less toxic than 5-FU to hematopoietic stem cells (3) . In oraler to better understand the differences in the toxicological properties of Ft and 5-FU, a study was made of the effects of equimolar doses of the two drugs on in vim, DNA synthesis in the small intestine of the rat . In addition, the in vivo uptake and metabolic disposition of the two drugs in the intestine was studied . The aesults of these investigations indicate that the difference in cytotoxic activity between Ft and 5-FU may be related to the more complete conversion of the latter drug into fluorouracil-containing nucleotides by normal proliferating host tissues .
1363
1364
Sffact of Ftorafur aad 5-Fluorouracil
Vol. 17, No . 9
Methods Male, Sprague-Dawley rats were given intravenous injections of Ft or 5-FU dissolved in 0 .2 M sodium bicarbonate/carbonate, pH 9 .5 . Control anlmala receivéd an equal volume of the buffer alone . At various intervals thereafter they received an intravenous injection of either (6-3H) deoxyuridine (76 ~ Cükq, specific activity, 21 ~ Ci/G,i mole) or (methyl- 3H) thymidine (80 ~ Ci/kq ; specific activity, 40 u Ci/u mole) and were killed 20 or 10 minutes later, respectively . The small intestine was removed and placed in ice-cold 0 .996 NaCI . The intestines were then slit open and agitated to remove fecal material . DNA was extracted from homogenates of the cleaned intestines by the procedure of Munro and Fleck (4) . The DNA content of the extracts were determined colorimetrically according to the method of Schneider (5) with deoxyadenosine serving as a standard . Radioactivity present in the extracts was determined by liquid scintillation spectrometry . Counting efficiency was estimated by internal standardization . (2- 14 C) ftorafur was prepared by Dr . Leroy B . Townsend, University of Utah (6) . (2- 14 C) 5-fluorouracil was purchased from Caltomic . The radiochemical purity of both compounds was found to be > 9796 following separation by the thin layer chromatography procedure deacrlbed below . Acid soluble metabolites were extracted from the small intestine at 0 .5 and 4 hours following the intravenous injection of (2- 14 C) Ft (50 ~ Ci/kq; specific activity, 50 ~ C!/100 mg) or (2- 14C) 5-FU (48 ~ Ci/kg; specific activity, 48 ~ Cî/65 mq) . The intestines were homogenized in three volumes of ice-cold water and an equal volume of wld 0 .4N perchloric acid was added . The acid insoluble material was removed by centrifugation and the supernatant was saved . The sediment was washed twice with 0 .2N perchloric acid and the washings were added to the original supernatant. The combined supernatant was then adjusted to pH 7 with potassium hydroxide . After allowing the neutralized extract to remain in the cold for several hours, the insoluble potassium perchlorate was removed by centrifugation . The extract was concentrated by freeze-drying and the residue was dissolved in a small volume of water . The metabolües present in the neu~alized extract were separated on silica gel chromatography plates containing a fluorescent indicator (Chromar 7 GF ; Mallinckrodt) . The location of the metabolites was facilitated by the use of non labeled standards which were spotted on top of the radioactive samples . These standards were then located under U .V . light after the chromatogram was developed . In all cases a duplicate radioactive sample was spotted ahead of the solvent front to allow for the determination of recovery of radioactivity from the chromatogram . The solvent system employed consisted of ethyl acetate: methanol : conc . NH40H (75 :25:1, V/V/V) . The Rf of the metabolites were as follows : Ft = 0 .74; 5-FU 0 .58; 5-fluoro-2'-deoxyuridine (FUdR) = 0 .47; 5-fluorouridine (FUR) = 0 .25 . Nucleotides (5-fluoro-2'-deoxyuridine-5'-monophoaphate and 5-fluorouridine-5'- ' monophoaphate) remained at the origin . Following development, each spot was scraped off, eluted with water and counted by liquid scintillation spectrometry . Counting efficiency was estimated by internal standardization . Results and Disc ussion Figure 1 shows the effect of equimolar doses of Ft (100 mg/'kq) and 5-FU (65 mq/kq) on the in vive incorporation of (6-3H) deoxyuridine into the DNA of the small intestine . Ft produced a 30-5096 decrease, in the rate of incorporation at one-two hours after intravenous injection. In the subsequent two hours the
Effect of Ftorafur and 5-Fluorouracil
Vol . 17, No . 9
1365
ts~
ta~
2
HOURS AFTER LV DOSE
FIG . 1 Effect of ftorafur and 5-fluorouracil on the incorporation of (6-3H) deoxyuridine into intestinal DNA. Each symbol is the mean specific activity of the DNA extinct and must be multiplied by 100 to obtain the actual DPM/(1 mole deoxyriboae . The vertical bars show the S .E, of the wean . Numbers in parenthesis denote the number of animals per treatment group . Asterisks indicate that the value for the treatment group differed significantly (p ~ 0 .05) from the control group . O, control; / ftorafur (100 mg/kq) ; ~, ftorafur (300 mg/kq) ; rate of incorporation decreased tD about 3596 of the control value . Thereafter, deoxyuridine incorporation began to increase ao that by 12 hours the rate was 66% of the control value . In contrast, an equimolar dose of 5-FU produced a greater than 90% inhibition in the rate of deoxyuridinè incorporation within one hour . Moreover, this degree of inhibition was sustained for at least 12 hours . Fig . 1 also shows that the administration of Ft at a dose of 300 mg/kg produced effects similar to those observed with 5-FU . However, even at this high dose level, maximum inhibition of deoxyuridine incorporation was not attained until two hours after injection of the drug . 5-FU is believed to exert its antitumor and toxic effects after being converted ill, vi to 5-fluoro-2'-deoxyuridine-5'-monophosphate which is a potent inhibitor of thymidylate synthetase (7,8) . Since Fti is an analog of 5-FU, it we~ of irnerest to determine if the thymidylate synthetase reaction was specifically inhibited by this new drug . Accordingly, the effect of Ft {300 mg/kg) on the ~l.yjYS incorporation of (methyl- 3 H) thymidine into intestinal DNA was measured at two and
1366
8ffect of !`torafur sad S-L~luorouracil
Vol . 17, No . 9
four hours after injection of the drug . It will be rocalled from Fiq. 1 that deoxyuridine incorporation was maximally inhibited by this dose of Ft at both of these time periods . The data in Table 1 show that in contrast to its effects on deoxyuridine incorporation at two hours , Ft did not significantly inhibit thymidine inwrTABLE 1 Effect of Ftorafur and 5-FU on the Incorporation of Thymidine into DNA of Rat Small Intestine l2PM/k M~e Deoxytibos~a
~I$. Control Ft(300 mg/kq) S-FU(65 mq/kq)
1868 _+ 398 (4) 1768 _+ 290 (4) 2193 + 259 (3)
1853 ± 134 (3) 3256 ± 323 (5) 2983 + 670 (4)
aMean ± S .E . The numbers in parenthesis rofer to the number of animals per treatment group . potation into DNA . At four hours a stimulation of thymidine incorporation was observed . This most likely reflected a decrease in the endogenous thymidine nucleotide pools which would oth®rwise be available to dilute the radioacttve thymidine infected into the rats . Table 1 also shows that similar rosults were obtained after infection of 5-FU and thus indicates that both drugs sham at least one common biochemical site of action . Since the observed differoncea in the effects of equimolar doses of Ft and 5-FU on deoxyuridine incorporation into DNA (Fig . 1) might have been rolated to differonces in the metabolic disposition of the two drugs, a study was made of the incorporation of radioactivity labeled Ft and 5-FU into the acid-soluble fraction of the small intestine . The data in Table 2 show that the fatal concentration of each drug and its metabolites were nearly equivalent at 30 minutes after the infection of equimolar doses of the two drugs . Ap~oximately 6596 of the total amount present after the infection of Ft was accounted for by the unmetabolized parent compound . Leas than 2096 of the drug had been converted to fluorouracil-containing nucleosides and nucleotides . In contrast, less than 259K of the total drug equivalents present in the extracts from rats given 5-FU was due m the unmetabolized drug . Furthermoro, fluorouracil-containing nucleotides accounted for nearly S09I6 of the total radioactivity present. In absolute terms , between throe and four times as much 5-FU was converted to nucleotides in the small intestine within 30 minutes after infection of equimolar doses of the two drugs . At four hours after the infection of Ft little change was observed in either the total concentration of drug equivalents or the distribution of metabolites . In contrast, the total concentration of drug equivalents from 5-FU was about half of that found at 30 minutes . However, fluorouracil-containing nucleotides still accounted for appraximaiely 5096 of the total radioactivity present . Theroforo, in absolute terms, the concentration of these nucleotides was about twice as great following 5-FU than after the infection of an equivalent amount of Ft . The prosent studies indicate that the reported lower toxicity of Ft as compared to 5-FU may be related to a relatively slow rate of conversion of the former drug into fluorouracil-containing nucleotides by normal proliferating hoot
Vol.
17,
No . 9
Effect of Ftorafur an8 5-Fluorouracil
1367
TABLE 2 Thin Layer Chromatography of Acid-Soluble Metabolites in Small Intestines METABOLITE CONCENTRATION (n mole/gm) Q" 5 hr
5-FU 213
Ft 195
4 t~
5-FU 97
TOTAL
Ft 230
Ft FU FUdR FUR Nucleotides
150 18 5 2 30
50 17 11 103
157 8 2 2 25
22 5 7 51
Recovery(%)
89
85
100
89
Pairs of rats received an intravenous injection of either (2- 14 C) ftomfur (50 u Ci/kg ; specific activity 50 ~ Ci/100 mg) or (2-14C) 5-fluorouridine (48 ~ Ci/kg; specific activity 48 ~t Ci/65 mg) . The rats were killed at 0 .5 or 4 hours thereafter and the acidsoluble metabolites were extracted and separated on silica gel plates as described in the text . Each value is the mean of a single pair of rata . tissues . 5-FU is believed to inhibit DNA synthesis and exert its cytotoxic effects only after it has been converted to 5-fluoro-2'-deaucyuridine-5'-monophoaphate . This nucleotide is a potent inhibitor of the enzyme, thymidglate aynthetase (7,8) . The more rapid and marked inhibition of deoxyuridine incorporation into the DNA of the small intestine after the administration of 5-FU as compared to Ft (Fig . 1) is aonsistant with the more complete conversion of the former drug to the nucleotide level ~ vivo (Table 2 ) . ~knowled2ement This work was supported by United States Public Health Service Grant CA 13238 . ~efe~ences 1 . S . A . H111er, R . A. Spuk , and M . Y. Lidak , Dokl . Adad . Nauk . (USSR), 176, 332-335 (1967) . 2 . N . G . Blokhina, E . K . Vozny and A . M . Garin, Cancer, 30, 390-392 (1972) . 3 . I. Hrsak, and S . Pavicic, Biomedicine , 21, 164-67 (1974) . 4 . H . N . Munro, and A . Fleck, Meth . Biochem . Anal ., 14, 112-176 (1966) . 5 . W. C . Schneider, Meth . Enzvmol., 3, 680-684 (1957) . 6 . R. A . Earl and L . B . Townsend, T. Heterocvcl . Chem ., 9, 1141-1143 (1972) . 7 . I. U . Hartmann, and C . Heidelberger, T . Biol . Chem ., 23fi, 2006-3013 (1961) . 8 . P . Reyes, and C . Heidelberger, Mol. Pharmacol., 1, 14-30 (1965) .