[CANCER RESEARCH 45, 5405-5412, November 1985]

Enzymes of Uracil Catabolism in Normal and Neoplastic Human Tissues1/

Fardos N. M. Naguib,2 Mahmoud H. el Kouni, and Sungman Cha

Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT

Enzymes of the pyrimidine base catabolism, dihydrouracil de-

hydrogenase (EC 1.3.1.2), dihydropyrimidinase (EC 3.5.2.2), and/3-ureidopropionase (EC 3.5.1.6) were compared in the cytosolic

extract of several normal and neoplastic human tissues. Theactivity was measured by following the catabolism of [6-14C]-

uracil to dihydrouracil, carbamyl-/3-alanine, and /3-alanine. Sub

strate inhibition, hysteresis, allosterism, and the lack of dihydropyrimidinase are pointed out as special problems in assayingenzymes of pyrimidine degradation. The activity of dihydrouracildehydrogenase has been demonstrated in several human extra-hepatic tissues and tumors. The enzyme is rate limiting in extra-

hepatic solid tumors but not in their normal counterparts. Someof these solid tumors contain greater amounts of activity thando their normal equivalents, which encourages the use of inhibitors of this enzyme in conjunction with treatment of these tumorsby 5-f luorouracil. Because of the lack of a pattern in dihydrouracil

dehydrogenase activity between tumors and normal tissues, theenzyme is not a good marker for tumorigenicity. Dihydropyrimidinase, on the other hand, is highly active in all solid tumorsstudied but not in their normal counterparts; therefore, we suggest that dihydropyrimidinase can serve as a good marker oftumorigenicity as well as a target for cancer chemotherapy ofhuman solid tumors.

INTRODUCTION

In mammalian systems the pathway for the catabolism ofuracil, thymine, and their analogues (Chart 1) is via degradativereduction by which the pyrimidine ring is first reduced at positions5 and 6 with hydrogen from NADPH, then opened betweenpositions 3 and 4, and finally split between position 1 and 2 toyield the corresponding <•>'amino acid, carbon dioxide, and am

monia.Dihydrouracil dehydrogenase (5,6-dihydrouracil: NADP+ oxi-

doreductase, EC 1.3.1.2) is the first and purportedly rate limitingenzyme of this chain of three reactions. It is responsible for thereversible reduction of both uracil and thymine to dihydrouraciland dihydrothymine, respectively (1-4). This enzyme is also

responsible for the breakdown of the widely used antineoplasticagent 5-fluorouracil, and the radiosensitizing agents 5-bromo-and 5-iodouracil, thereby limiting their therapeutic effectiveness.Cytosine and its analogues are not substrates for this enzymeand have to be converted to uracil before entering the reductivedegradation pathway. Most reports locate dihydrouracil dehydrogenase activity in liver cytosol (2, 5-9). However, an additional

dehydrogenase activity in the paniculate fraction has also beenreported (10,11 ). Dihydrouracil dehydrogenase has been purified

'Supported by Grants CA-31706 and CA-13943 awarded by the NationalCancer Institute, Department of Health and Human Services, and Grant CH-136

from the American Cancer Society.2To whom requests for reprints should be addressed.

Received 3/18/85; revised 6/9/85; accepted 7/22/85.

to homogeneity from rat liver cytosol (9).Dihydropyrimidinase (5,6-dihydropyrimidine amidohydrolase,

EC 3.5.2.2) is the second enzyme of pyrimidine base degradation. It is responsible for the reversible hydrolytic ring opening ofdihydrouracil and dihydrothymine and is located in the cytosolfraction (12-15).

/3-Ureidopropionase (A/-carbamoyl-j8-alanine amidohydrolase,

EC 3.5.1.6) is the last of the pyrimidine degradative enzymes. Itsplits the /3-ureidopropionic acid (carbamyl-ß-alanine) or /3-urei-

doisobutyric acid formed by dihydropyrimidinase from dihydrouracil or dihydrothymine into 0-alanine or /9-aminoisobutyric acid,ammonia, and carbon dioxide (Chart 1). /3-Ureidopropionase is

also located in the cytosol (14, 16, 17). The reaction catalyzedby this enzyme is irreversible.

Reports on dihydrouracil dehydrogenase activity in the variousextrahepatic tissues are contradictory. The kidney was reportedto be the only extrahepatic tissue to contain dihydrouracil dehydrogenase (4,18-20). However, this activity was later reported

to be present in rat thymus, intestinal mucosa, spleen, kidney,brain cortex, skeletal muscles, heart, lung, stomach, and bonemarrow (21,22); in mouse colon and colon tumors (23); in humankidney and kidney tumors (24); and in normal and neoplastichuman colon, lung, and stomach (25). The early discrepancies inthe literature could be attributable to the assay conditions usedto determine enzyme activity. Higley and Buttery (26) reportedthat none of the standard assays developed by Grisolla andCardoso (5), Smith and Yamada (10), or Fritzson (6) provedsatisfactory.

In the present study while determining dihydrouracil dehydrogenase activity in normal and neoplastic tissues from differenthuman organs, we have initially experienced some difficulties inobtaining optimal conditions for the measurements of this enzyme activity. We found that substrate inhibition by uracil, enzyme hysteresis, and allosterism were the main factors behindthis difficulty. This prompted us to study the kinetic parametersof this enzyme in the cytosol of various tissues and organisms.We herein demonstrate that the enzyme from the varioussources studied displayed substrate inhibition by uracil, and thatunder appropriate assay conditions, the activity of dihydrouracildehydrogenase can be detected in various normal and neoplastichuman tissues. However, no definitive pattern in the relationshipbetween enzyme activity in the normal and in correspondingneoplastic tissues could be established. Dihydropyrimidinase, onthe other hand, was absent from or showed little activity innormal extrahepatic tissues as well as all neoplastic lymphoidtissues tested. In contrast all solid tumours tested had appreciable dihydropyrimidinase activity. A preliminary report has beenpresented (27).

MATERIALS AND METHODS

Chemicals. [6-"C]Uracil (56 mCi/mmol) was purchased from MoravekBiochemicals, Brea, CA; [6-14C]5-fluorouracil (55 mCi/mmol) was from

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ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES

+ NH, -. CO,

K = H

R=CH,

R= F

UKACIl

THVMIHE

flUOROURACIL

OIHYDROURACIt

DIHYDItOTHVMINE

DIHYMOFLUOROUKACIL

«-tWEIOOntOPIOIUTE

0- UMEIDOIKMUTVKATf

«-FLUORO-«-UREIOOFItOPIONATE

.-•AlANI«

«-AMINOIKWUTYRATE

.. HUORO s ALAMINÕ

Chart 1. Pathway of pyrimidine base catabolism in mammalian systems.

Amersham Corp., Arlington Heights, IL; uracil, dihydrouracil, carbamyl-

0-alanine, /3-alanine, NADPH, and ninhydrin, were from the Sigma Chem

ical Co., St. Louis, MO; Polygram CEL 300 UN/UMand silica gel G/UVuMTLC3 plates were from Brinkmann Instrument Co., Westbury, NJ; Om-

nifluor was from New England Nuclear, Boston, MA; dimethylamino-

benzaldehyde was from Aldhch Chemical Co., Milwaukee, Wl, and allother chemicals were from Fisher Scientific Co., Boston, MA.

Normal Tissues. Normal human organs were obtained from autopsies, peripheral blood lymphocytes were prepared from the blood ofvolunteers by the method of Boyum (28), and normal murine organswere obtained from Swiss albino (CD-1) mice or Lewis rats (Charles

River Breeding Laboratories, Wilmington, MA). Mice were killed bycervical dislocation and rats by decapitation. The organs were washedwith ice-cold normal saline (0.9% NaCI solution) before homogenization.

Solid Tumors. Carcinomas of the colon, established from biopsies,were grown as xenografts in nude mice: DLD-1, a carcinoma of the

sigmoid colon, morphologically heterogeneous, varying from moderatelyto poorly differentiated (29); clone A, a subclone of DLD-1, producing

poorly differentiated adenocarcinomas (29); clone D, another subcloneof DLD-1, producing moderately differentiated adenocarcinomas (29);DLD-2, a well differentiated adenocarcinoma of the sigmoid colon; HCT-

15, a moderately well differentiated adenocarcinoma of the sigmoid colon(29); HOT-3, a metastasis to the ovary of a patient with a sigmoid colon

primary cancer, producing well differentiated adenocarcinomas (30);lntob-3, derived from a metastasis to the omentum of a patient with a

colorectal primary and producing well differentiated adenocarcinomas;and OM-1, derived from a metastasis to the omentum of the samepatient that gave rise to HOT-3, also producing well differentiated ad

enocarcinomas (30).DAN, a carcinoma of the pancreas, was grown in culture. This cell line

was derived from a metastasis to the liver of a patient with pancreaticprimary cancer. Histologically the specimen is described as a squamouscell carcinoma (31 ).

Carcinomas of the pancreas, derived from liver biopsies from patientswith primary cancer metastatic to the liver, were grown as xenografts innude mice; RWP-1, a liver metastasis from a primary duct cell of thehead of the pancreas; and RWP-2, a moderately well differentiated duct

cell adenocarcinoma of pancreatic origin (32).LX-1, a carcinoma of the lung, was grown in culture. This cell line was

established from a metastasis to the arm of a patient with an inoperableprimary oat cell carcinoma of the lung. Histologically the specimen isdescribed as a nodule of poorly differentiated carcinomas (33).

HLN-3, a salivary gland carcinoma, was grown as xenografts in nude

mice. This carcinoma was established by Dr. D. L. Dexter and colleaguesfrom a cervical lymph node metastasis following appearance of anunspecified salivary gland primary site.

HST-2, a carcinoma of the stomach, was grown as xenografts in nude

mice. This carcinoma was established by Dr. D. L. Dexter and colleaguesfrom a portion of the stomach of a patient with a moderately welldifferentiated adenocarcinoma.

Neoplastia Hematopoietic Tissues. Leukemic cell lines were grownin culture: ARH-77, a B-lymphoblast line established from the peripheral

3The abbreviation used is: TLC, thin layer chromatography.

blood of a patient with immunoglobulin plasma cell leukemia (34); K-562.

an undifferentiated blast cell line established from the pleural fluid of apatient with chronic myelocytic leukemia (35); KG-1, a mixture of pre

dominantly myeloblasts and promyelocytes derived from the bone marrow of a patient with erythroleukemia (36); and HL-60, a promyelocytic

cell line established from the peripheral blood of a patient with acutepromyelocytic leukemia (37).

RWLy-1, is a lymphoma line established and characterized by Wie-mann." It was isolated from the pleural effusion of a patient with mixedhistiocytic-lymphocyticnon-Hodgkin's lymphoma. In tissue culture, about

50% of RWLy-1 cells have the morphological appearance of small

malignant lymphocytes and the other 50% have the appearance of largehistiocytes. Cell surface IgM can be detected by immunofluorescence ingreater than 70% of the cells. RWLy-1 is an Epstein-Barr virus-free cell

line.Preparation of Extracts. Cells in suspension were collected by cen-

trifugation. The cells were washed by resuspending in the homogenization buffer (20 mw potassium phosphate (pH 8) containing 1 rriM EDTAand 1 mw mercaptoethanol). After washing twice the pellet was homogenized in 2 volumes of buffer, using a polytron homogenizer (Brinkmann).The homogenate was then centrifugea at 105,000 x g for 1 h at 4°C.

The supernatant fluid (cytosol) was collected and used as the source ofenzyme. Other tissues were homogenized (1:2-3, w/v) in the homoge

nization buffer and the cytosol was prepared as described for neoplasticcell lines. When used for kinetic parameter estimations, HL-60 cells weretreated for 7 days with the maturational agent W,A/-dimethylformamide

(10.8%) before extracting the cytosolic fluid. This treatment was necessary to increase the level of dihydrouracil dehydrogenase activity some10-fold (38).

Enzyme Assays. Dihydrouracil dehydrogenase activity was determined by measuring the sum of the products, dihydrouracil, carbamyl-j3-alanine, and 0-alanine, formed from [6-14C]uracil. The standard reaction

mixture contained 10 mM potassium phosphate (pH 8), 0.5 mw EDTA,0.5 mW mercaptoethanol, 2 mw dithiothreitol, 5 mw MgCI2, 25 //M[6-'4C]uracil (56 mCi/mmol), 100 MM NADPH, and 25 n\ cytosol (5 mg

protein/ml) in a final volume of 50 n\. Incubations were carried out at37°Cfor 5 to 30 min, except where stated otherwise. The reaction was

terminated by immersing the reaction tubes (1-ml Eppendorf tubes) in a

boiling waterbath for 1 min, the reaction tubes were then frozen at-20°C for at least 20 min before any further manipulations were under

taken. Proteins were removed by centrifugation and 5 ¡Aof the supernatant fluid were spotted on cellulose TLC plates which were prespottedwith 5 /il of a standard mixture of 10 mw uracil, carbamyl-,¡-alanine, ß-

alanine, and 25 mw dihydrouracil. The plates were then developedovernight in the top phase of a mixture of n-butanol:water:ammonium

hydroxide (90:45:15, v/v/v). Uracil was identified by UV quenching and/3-alanine by spraying with 0.2% ninhydrin in 95% ethanol. Dihydrouraciland carbamyl-|8-alanine were visualized by dyeing with 5% dimethylami-

nobenzaldehyde in 50% ethanol:1 N HCI, after dihydrouracil has beenhydrolyzed to carbamyl-/J-alanine by spraying with 0.5 N KOH in 50%

ethanol. R( values for dihydrouracil, uracil, and carbamyl-/3-alanine plus/3-alanine were 0.46, 0.23, and 0.09, respectively. Spots were cut out

4 M. C. Wiemann. details to be published elsewhere.

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ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES

and placed in vials subsequently filled with 20 ml of Omnifluor-basedscintillant. Radioactivity was counted in a Packard Tri-Carb Model 460scintillation counter. The presence of dihydropyrimidinase and /3-ureido-propionase activities was inferred from the formation of carbamyl-/3-alanine and 0-alanine, respectively, in the dihydrouracil dehydrogenaseassay described above. On cellulose TLC plates, carbamyl-j-alanine and/3-alanine do not separate from one another. Therefore they were sepa

rated from each other on silica gel TLC plates. The plates were developedin chloroform:methanol:formic acid (65:18:1, v/v/v). The R( values foruracil, carbamyl-.i-alanine. dihydrouracil, and .¡-alanine in this system

were 0.66, 0.55, 0.24, and 0.13, respectively. This system was not usedroutinely for the separation of all uracil catabolites because of the frequentdifficulty in visualizing dihydrouracil after dyeing with dimethylaminobenz-

aldehyde.Protein Estimation. Protein concentrations were determined by the

method of Bradford (39) as described by the Bio-Rad Laboratories (40),using bovine ^ -globulin as a standard.

RESULTS

Dihydrouracil Dehydrogenase Activity versus Time andAmountof Enzyme. Chart 2 showsthat inmouseliver,withinacertain limit of incubation time and protein concentration, dihydrouracil dehydrogenase activity was linear with respect to timeafter an initial lag and to amount of enzyme. The time lag is adistinctive characteristic of hysteresis (41). Similar results wereobtained with the promyelocytic line HL-60 and human and ratlivers (data not shown). In contrast in peripheral blood lymphocytes dihydrouracil dehydrogenase activity (120 pmol/min/mg)was strictly linear with respect to time from 0-30 min.

Substrate Inhibition by Uracil and NADPH. Charts 3 and 4show that high concentrations of uracil inhibited dihydrouracildehydrogenase from human and rat livers, respectively. Inhibitionby high concentrations of uracil was also detected with extractsfrom the promyelocytic cell line HL-60, DAN, LX-1, and mouseliver (data not shown). Chart 5 shows that high concentrationsof the cofactor NADPH also inhibited dihydrouracil dehydrogenase from HL-60 enzyme. Moreover the concentration of either

100x

80

60

40

20

15 mg

20 40TIME, min

60 5 IOP ROTEI N, mg/ml

Chart 2. Activity of dihydrouracil dehydrogenase from mouse liver. Assay conditions are as described in "Materials and Methods." Points, mean from three

determinations. A, activity with time shows hysteresis (time lag); B. linearity withamount of enzyme.

0.03

X9E•v^c

10.02o

0.01

0.01 0.02 0.03l/(Urocil), juM-'

Chart 3. Activity of dihydrouracil dehydrogenase in human liver cytosol. Plot of1/velocity versus 1/uracil at two fixed concentrations of NADPH of 100 JIM(•)and500 UM(•)•The reaction mixture was incubated at 37°Cfor 10 min. Points, mean

from three determinations. The kinetic parameters estimated from the straight lineportion of each curve were: Km = -580 and 14 ^M uracil, respectively; and V„»=-513 and 155 pmol/min/mg protein, respectively.

o.o2r

'SEvC

E>.20.01

0.1

1/lUrocil),

Chart 4. Activity of dihydrouracil dehydrogenase in rat liver cytosol. Rot of 1/velocity versus 1/uracil at a fixed concentration of NADPH of 100 »u.The reactionmixture was incubated at 37°Cfor 5 min. Points, mean from three determinations.

The kinetic parameters estimated from the straight line portion of the curve were:Km = 6 UM uracil and Vâ„¢,= 242 pmol/min/mg.

substrate or cofactor mutually influenced the concentration atwhich the other became inhibitory. With a higher concentrationof the cofactor (1 mMor above), inhibition was observed at lowersubstrate concentrations. Table 1 also lists the concentrationsof uracil and NADPH at which optimal rates were observed, aswell as the conditions under which these estimates were obtained.

Determination of the Apparent Km and Vm„in VariousTissues. It was not possibleto measuredihydrouracildehydrogenase activity in various tissues at one optimal substrate con-

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ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES

centration. Indeed the standard assay conditions chosen wereslightly inhibitory to the HL-60 enzyme; nonetheless these con

ditions were adopted because they were appropriate for theenzymes from most other tissues tested. The linear portion onthe double reciprocal plot was used to determine the apparentKmvalues for uracil and NADPH in the different tissues presentedin Table 1, along with other kinetic parameters. The data shouldbe taken as approximate values, since the enzyme used wasnot purified. Nonetheless the results in Table 1 show that the Kmvalues for uracil [5.8 ±0.7 (SD) fM] and NADPH [9.6 ±0.6 UM]estimated for rat liver enzyme are compatible with the Kmvaluesreported for uracil [1.8 ^M] and NADPH [11 ^M], with purified ratliver enzyme (9). The differences in estimated Kmvalues betweenvarious tissues suggest that, at least in humans, there may existmore than one isozymic form of this enzyme.

Chart 3 shows that in the human liver extract, NADPH con-

0.05

I/(NADPH), >uM-«Charts. Activity of dihydrouracil dehydrogenase in HL-60. Ptot of 1/velocity

versus 1/NADPH at a fixed concentration of uracilof 100 MM.The reaction mixturewas incubated at 37°Cfor 5 min. Points, mean from three determinations. Thekinetic parameters estimated from the straight line portion of the curve were: Km=2 MMNADPHand V™,= 45 pmol/min/mg.

centration had to be raised to 0.5 mu (approximately 5.5 x theestimated K,,,value for NADPH) in order to obtain a Kmvalue foruracil, otherwise the double reciprocal plot gave a negative Kmvalue. Negative Km values were also observed with humanplatelets and mouse kidney extracts. These results indicate thatdihydrouracil dehydrogenase from these sources is of the allo-steric type. The depletion of NADPH by other liver dehydrogen-

ases has been assessed and ruled out.Dihydrouracil Dehydrogenase Activity in Normal and Neo-

plastic Human Tissues. Table 2 shows the levels of dihydrouracil dehydrogenase activity in various normal and neoplastichuman tissues. Of the normal tissues, peripheral blood lymphocytes and liver contained the highest activities. Of the neoplasms,both the leukemic cell line, KG-1, and the pancreatic carcinomas,DAN and RWP-1, had relatively high levels of activity, equivalent

to that of normal liver, yet lower than that of normal lymphocytes.When compared with their normal counterparts no significantdifference (P > 0.05) was observed between normal intestinalmucosa and colon tumors or between normal lung and the lungtumor LX-1. In contrast the activity of the pancreatic tumorsDAN and RWP-1 were significantly higher (P < 0.01) than that

of the normal pancreas. Therefore we could not assign anydistinctive pattern to enzyme activity in tumors relative to thecorresponding normal tissues.

Formation of Dihydrouracil, Carbamyl-0-alanine and 0-Ala-nine from "C-Uracil by Various Human Tissues and Organs.

Table 2 also shows the relative amounts of dihydrouracil, car-bamyl-/3-alanine, and 0-alanine formed from uracil by the cytosolic

extract of different human tissues and organs. In normal liver90% or more of the products formed were carbamyl-0-alarïmeand n'-alanine, while in normal lung and pancreas only 5-8% of

the total product formed was carbamyl-j8-alanine and /3-alanine.

In normal peripheral blood lymphocytes and intestinal mucosa,the catabolism of uracil could not be detected beyond dihydrouracil, indicating a very low or a lack of dihydropyrimidinase activity.As for the neoplasms, all of the human leukemic lines tested aswell as the lymphoma line RWLy-1 accumulated their products

as dihydrouracil. In contrast all solid tumours catabolized uracilto carbamyl-(8-alanine and /3-alanine. These results indicate that

dihydropyrimidinase is highly active in all of the solid tumorstested, unlike their normal counterparts. Furthermore the presentresults also show that in solid neoplasms, with the exception ofthe pancreatic tumor RWP-1, the pattern of uracil catabolism

Table 1Kinetic parameters of dihydrouracil dehydrogenasefrom various tissues

Kmvalues for uracil were determined using 100 MMNADPH and uracil concentrations ranging from 4 to 100 MM.Kmvalues for NADPH were obtained using 25 MMuracil and NADPH concentrations ranging from 4 to 50 MM.

Apparent Km(uracil)(MM)V„»(pmol/min/mg)V„^K„[Uracil]

(MMat optimumrate)Apparent

Km(NADPH)(MM)V™,(pmol/min/mg)V„,/Km[NADPH]

(MMat optimum rate)Human

HL-601.1±0.2*39.3

±1.735.7501.7±0.1C45.2

±0.626.15Human

lymphocytes18.7

±1.8137.3±4.97.3>80ND"NDNDNDHuman

liver14.3±1.46155.1

±3.010.817587

.4 ±21.738.7±7.00.4NDRat

liver5.8

±0.7241.9±10.141.7409.6

±0.6220.6±3.923.080Mouse

liver9.3

±0.9724.7±18.978.3>100900

±1.90016,600±33,10018.2ND

" Mean ±SD.6 Determinedat 500 MMNADPH.' Determinedat 100 MMuracil" ND, not determined.

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ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES

Table 2Activity of dihydrouracil dehydrogenase and distribution of the products formed from uracil by the extracts of various human tissues

Assay conditions and tissue sources are those described in "Materials and Methods.'

Spécifieactivity(pmol/min/mgprotein)TissuesNormalLiverPancreasLungIntestinal

mucosaLymphocytesNeoplasmsSolid

tumorsColonDLD-1Clone

ACloneDDLD-2HCT-15HOT-3lntob-3OM-1LungLX-1PancreasDANRWP-1RWP-2Salivary

glandHLN-3StomachHST-2Hematopoietic

tumorsLeukemiccellsARH-77K-563KG-1HL-60Lymphoma

cellsRWLy-1"

N, number of samplestested.*Counted with car bamyl-, i-alanmeMean

±SO21

.5±6.04.3±1.35.2

±2.87.1±6.9106.3±21.53.8

±0.42.9±1.33.7

±0.40.9±0.22.4±0.52.8±2.81.9

±1.64.0±3.215.4

±12.124.3

±5.3e16.3±2.1°4.1

±0.15.1

±1.84.0

±3.33.5

±2.37.8±2.625.4±6.71.9

±0.31.2

±0.2N*322222222222233222233333Coefficient

ofvariationwithin

samples0.01-0.040.18-0.380.04-0.090.05-0.180.01-0.020.09-0.220.10-0.470.11-0.150.10-0.850.05-0.070.14-0.500.10-0.370.36-0.470.03-0.300.01-0.160.01-0.090.07-0.170.07-0.150.36-0.470.09-0.300.03-0.260.02-0.050.12-0.500.16-1.30Dihydrouracil11929510010041891066272803385745100100100100100%

of productformedCarbamyl-0-alanine47850066444846555636385231958565400000.(•AlanmeI2bb0030384344393837354835635404100000c

Significantly (P < 0.01) different from normal pancreas.

resembles that of the liver, in that the major product formed (70-100%) was carbamyl-ii-alanine and /8-alanine as opposed to theirnormal equivalents, where the major product formed (90-100%)

was dihydrouracil.Activity toward Fluorouracil. Table 3 shows the activity of

dihydrouracil dehydrogenase from various tissues toward uracilor 5-fluorouracil as well as the distribution of the 5-fluorouracilcatabolites. The results indicate that in all tissues tested, 5-

fluorouracil is a better substrate for dihydrouracil dehydrogenasethan uracil. Dihydrofluorouracil too may be a better substrate fordihydropyrimidinase than dihydrouracil. Table 3 also shows differences in the ratio of dehydrogenase activity toward 5-fluo

rouracil relative to that toward uracil in human liver and peripheralblood lymphocytes, which supports our suggestion that thelymphocyte enzyme may be a different isozyme form from thatof the liver. Similar results were observed with the enzymes fromthe liver and kidney of the rat (Table 3).

DISCUSSION

The present results demonstrate the occurrence of dihydrouracil dehydrogenase activity in all the tissues tested, in agreementwith the results of some investigators (21-25). This is contrary

to the results of others (4, 18-20) who could not demonstrate

dihydrouracil dehydrogenase activity except in livers and kidneysof various animals. Our results indicate that the failure of thoseinvestigators (4, 18-20) to demonstrate this activity in extrahe-

patic and extrarenai tissues could be due to complications intheir assay system arising from substrate inhibition, hysteresis,allosterism, and the absence of dihydropyrimidinase from thecatabolic pathway of pyrimidine bases.

Dihydrouracil dehydrogenase from various tissues and organsis inhibited by both substrates, uracil and NADPH (Charts 3-5).

We have reported substrate inhibition by uracil (27), and Queeneref al. (21) observed substrato inhibition by NADPH. Pero ef al.(42) showed the same phenomenon occurring with thymine inhuman platelet extracts. Inhibition of dihydrouracil dehydrogenase by its substrates appears to be a general characteristic ofthis enzyme, although the substrate concentration at whichinhibition occurs may differ from one tissue or animal to theother. For example we found that optimal substrate concentrations established for extracts from mouse liver ([uracil] = 0.25mw; [NADPH] = 3 mw) were inhibitory for human extrahepatic

tissues. Whether or not substrate inhibition plays a role in theregulation of pyrimidine base catabolism in vivo remains to bedetermined.

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TablesActivity of dihydrouracil dehydrogenase toward uracil or 5-fluorouracil and distribution of the products formed from 5-fluorouracil

Assay conditions are those described in "Materials and Methods."

Substrate(pmol/min/mg protein)

Tissue Uracil 5-Fluorouracil Ratio

% of product formed from 5-fluorouracil

FluoroureidopropionateDihydrofluorouracil + fluoro-.i-alanine

Human liverHuman lymphocytesRat liverRat kidney26.4

±5.06

88.8 ±6.295.9 ±6.929.1 ±3.7110.3

±2.5216.7 ±6.2400.0 ±2.945.9 ±7.04.2

2.44.21.51

1000299

0100

98aRatio of enzyme activity toward 5-fluorouracil to that towarduracil.hMoan -*- Cn

Chart 2A shows that the mouse liver enzyme definitely displayed a lag period, indicative of hysteresis (41). Similar resultswere observed with enzymes from HL-60 and human and rat

livers (data not shown). However, the peripheral blood lymphocyte enzyme did not exhibit any such time lag, and among allthe tissues tested, only human lymphocytes lacked this characteristic. Therefore it may be considered that, in humans, thelymphocyte has an isozyme different from that of the liver.

In addition to hysteresis some human tissues, e.g., liver andplatelets but not lymphocytes, showed negative Km values onregular double reciprocal plots. This is indicative of allosterism,/.e., a sigmoid curve on the velocity versus concentration plot. Inhuman liver this allosterism disappears when the concentrationof NADPH is increased to 0.5 ÕTIM(Chart 3). The absence ofallosterism from the lymphocyte and its presence in the liverenzyme support our contention that dihydrouracil dehydrogenase may have more than one isozymic form. This contention isfurther supported by the observed differences in Km values forthe enzyme from various tissues (Table 1) as well as in the ratioof the activity toward 5-fluorouracil to that toward uracil between

human liver and lymphocytes, and between rat liver and kidney(Table 3). The existence of two isoenzymes for dihydrouracildehydrogenase was reported in rat liver cytosol (8).

It could be argued that our results on enzyme activities inorgans obtained from autopsies may not represent the actualactivity in fresh tissues. However, the enzymes of pyrimidinebase catabolism from human liver and lymphocytes were quitestable when stored at -10°C, or after repeated freezing and

thawing, for over 1 week. These enzymes were also stable whenstored at -70°C for several months. Similar remarks were made

for the enzyme from rat liver (43). Furthermore we found thatthe activity toward 5-fluorouracil in human liver autopsy speci

mens (14.1 nmol/min/g tissue) is comparable to that (16.9 ±2.5nmol/min/g tissue) reported in liver biopsies obtained from patients (25). In addition hysteresis and allosterism have been alsoobserved in nonautopsy organs, e.g., mouse liver and kidney,respectively. We therefore believe that, although human liverswere obtained from autopsies and as such could have hadaltered enzymatic activity, this possibility is quite unlikely.

It was reported that dihydrouracil dehydrogenase is the ratelimiting enzyme for pyrimidine base catabolism in rat liver (2-4,43). On the other hand dihydropyrimidinase was suggested tobe the rate limiting enzyme in rat hepatocytes (44, 45) and ß-ureidopropionase in mouse liver (14). Our present results (Table2) suggest that dihydrouracil dehydrogenase is the rate limitingenzyme in human liver and all solid tumors tested with theexception of the pancreatic carcinoma RWP-1. In contrast dihy

dropyrimidinase may be the rate limiting enzyme in human extra-hepatic tissues and hematopoietic neoplasms, as 90% or moreof the total product formed was dihydrouracil.

Dihydrouracil dehydrogenase degrades the widely used anti-cancer agent 5-fluorouracil more efficiently than it does the

natural substrates uracil and thymine (Table 3, Refs. 9, 22, 46,and 47). Nevertheless few inhibitors for this enzyme are presentlyavailable, even though it has been reported that the antitumoractivity of 5-fluorouracil can be potentiated by 5-cyanouracil (48)and 5-diazouracil (49). The reason that (»administration of dihydrouracil dehydrogenase inhibitors with 5-fluorouracil has not

been popular is the fact that it was generally accepted thattumors lack or possess very little of this activity (50-52). Thisassumption was made on the basis of studies on mouse and rattumors (18,19, 22, 23, 53, 54) which may not represent humantumors. Dihydrouracil dehydrogenase is present in all of thehuman tumors studied (Table 2; Refs. 24 and 25). Furthermoreour results (Table 2) and those of others (24, 25) show that in allthe human tumors studied, with the exception of kidney (24) andliver (25) tumors, dihydrouracil dehydrogenase activity wasequivalent or higher in the tumors than in their normal counterparts. Maehera et al. (25) showed that human colon, stomach,and lung tumors had similar activity to that of normal tissues. Inaddition our results show that in certain tumors such as DAN,RWP-1, or KG-1, the activity is high and equivalent to that of

normal liver (Table 2). Therefore we suggest that active searchfor inhibitors of dihydrouracil dehydrogenase activity may beuseful in the treatment of, at least, these types of tumors with5-fluorouracil or similar compounds.

The catabolism of uracil did not proceed substantially beyonddihydrouracil in normal human pancreas, lung, intestinal mucosa,peripheral blood lymphocytes, all of the leukemic lines tested,and the lymphoma line RWLy-1 (Table 2). These results dem

onstrate the presence of little if any dihydropyrimidinase activityin these tissues. Similar results were reported with human peripheral blood platelets (42); rat brain extract (55); sliced orminced rat spleen, bone marrow, lymph node, adrenal, testis,thymus, lung, brain, heart muscle, skeletal muscle, skin, and afew tumors (13, 16, 56); and with perfused intestine, perfusedkidney and eviscerated preparations of the rat (57). ,8-Ureidopro-pionase was present in all tissues which had dihydropyrimidinaseactivity (Table 2).

The absence of dihydropyrimidinase may be the major factorfor the failure of some investigators (18-20) to detect dihydrour

acil dehydrogenase activity in extrahepatic tissues. These investigators were using the amount of 14CO2released from [2-14C]-

uracil as an estimate of dihydrouracil dehydrogenase activity in

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ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES

these tissues. In contrast we measured enzyme activity as theamount of 14C-labeled dihydrouracil, carbamyl-/J-alanine, and ß-alanine formed from [6-14C]uracil. This allowed us to detect a

lack of dihydropyrimidinase but not dihydrouracil dehydrogenasefrom certain tissues, when the accumulation of radioactive dihydrouracil but not carbamyl-(8-alanine and /3-alanine was ob

served. Had we measured dihydrouracil dehydrogenase activityas a function of the amount of CO2 released from [2-14C]uracil,

we would have reached the erroneous conclusion that thisenzyme is absent from extrahepatic tissues, since the absenceof dihydropyrimidinase from a tissue will not allow the formationof CO2, regardless of the presence of dihydrouracil dehydrogenase.

One of the most striking finding in this study is the reappearance or increase in dihydropyrimidinase activity in all the solidtumors tested when compared with normal tissues (Table 2). Infact a greater qualitative difference between the tumors and thenormal tissues is found in dihydropyrimidinase rather than indihydrouracil dehydrogenase activity (Table 2). This coincideswith our suggestion that in all normal human extrahepatic tissuestested, dihydropyrimidinase rather than dihydrouracil dehydrogenase is the rate limiting enzyme in the pyrimidine base cata-

bolic pathway. We therefore suggest that future studies bedirected at dihydropyrimidinase and at developing inhibitors ofthis enzyme. This suggestion is made more interesting by thefinding that dihydrofluorouracil contributes to the toxicity of 5-

fluorouracil, as the former persisted in blood circulation long afterthe latter had been cleared (58). Fluroro-/3-alanine, on the other

hand, had no toxic effect at all (58). This would suggest thatdihydrofluorouracil may function as a more stable depot form of5-fluorouracil. Furthermore the present study shows that in solid

tumors, in contrast to their normal counterparts, dihydrouracildehydrogenase, the enzyme responsible for the conversion ofdihydrofluorouracil to fluorouracil, is rate limiting. This indicatesthat dihydropyrimidinase has a higher activity than does dihydrouracil dehydrogenase in these tumors; hence it is importantto inhibit dihydropyrimidinase in order to increase the toxicity ofdihydrofluorouracil in such tumors.

In conclusion dihydropyrimidinase is highly active in all solidtumors studied but not in their normal counterparts; thereforewe suggest that dihydropyrimidinase can serve as a good markerof tumorigenicity as well as a target for cancer chemotherapy ofthe human solid tumors.

ACKNOWLEDGMENTS

We wish to thank Drs. F. W. Burgess, K. C. Agarwal, M. Y. Chu, D. L. Dexter,and M. C. Wiemann of Brown University and Roger Williams General Hospital,Providence,Rl, for providing the peripheralblood lymphocytes, platelets, tumor celllines, tumor xenografts, and autopsy materials used in this study. We also wish toextend our thanks to Ann Hollimanand Norma Messier for their excellent technicalassistance.

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