[CANCER RESEARCH 44,4955-4961, November 1984]

Catabolism of Exogenously Supplied Thymidine to Thymine and Dihydrothymine by Platelets in Human Peripheral Blood1

Ronald W. Pero,2 Desmond Johnson, and Anders Olsson

Wallenberg Laboratory, Biochemical and Genetic Ecotoxicology, University of Lund, Box 7031, S-220 07 Lund, Sweden [R W. P.. A. O.J, and Biochemistry Department, University College, Galway, Ireland [D. J.]

ABSTRACT The determination of UDS is particularly affected by exoge nously supplied dThd, because it is measured as the unsched The interference of platelets with the estimation of unsched uled incorporation of |3H|dThd into DMA at specific activities of uled DNA synthesis in human peripheral mononuclear leukocytes pHJdThd in the range of 10 to 30 Ci/mmol. Hence, the pHJdThd following genotoxic exposure was studied. A 96% reduction in concentration added to leukocyte cultures is quite low, which, in the unscheduled DNA synthesis value was achieved by incubat turn, would make this method unusually sensitive to any dThd ing [3H]thymidine with platelet-rich plasma for 5 hr at 37°.Using catabolic activity present in the blood. radioactive thymine-containing compounds, together with quan Earlier,we had found(13) that whole blood,or humanmono- titative analyses based on thin-layer and ion-exchange chroma- nuclearleukocytesculturedin the presenceof PRP, gave lower tographies, we have shown that thymidine was converted to UDSvaluesthanwholeblooddepletedof plateletsor leukocytes thymine which, in turn, was converted to dihydrothymine in culturedinthe presenceof PPP. Furthermore,itwas shownthat platelet-rich plasma. The enzymes responsible were separated this was not due to inhibitionof UDS by any of a numberof from platelet lysates by gel filtration and were identified as factors associatedwith platelet metabolism;e.g., cyclicAMP, thymidine phosphorylase and dihydrothymine dehydrogenase. cyclicGMP, sodiumarachidonate,or prostaglandinE2.On the The phosphorylase reversibly catalyzed the formation of thymine otherhand,plateletsareknownto possessdThdphosphorylase from thymidine and converted bromodeoxyuridine to bromoura- activity(6), and we have recentlyshown (12) dThd catabolism cil. The dehydrogenase reversibly catalyzed the interconversion by platelet-contaminatedleukocytecultures under conditions of thymine and dihydrothymine in a reaction dependent on which strongly reduced the calculated level of UDS. In our NADP(H), and it was inhibited by diazouradl and by thymine. continuationof this work, reportedbelow, we have more fully Nearly all the thymidine-catabolizing activity found in whole blood characterizedthe pathwayand enzymesof dThd catabolismin samples supplied exogenously with thymidine was accounted platelets.We have also shown that it is the catabolismwhich for by the platelets. Since most genetic toxicological tests that interferesso seriouslywith UDS measurements,and by dem use blood samples do not involve removing platelets from the onstratingthecatabolismofBrdUrd,we pointto the implications blood cell cultures, then it is concluded that precautions should for protocolsusingthatcompoundofcontaminationbyplatelets. be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods depend ent on thymidine pulses such as unscheduled DNA synthesis, or MATERIALS AND METHODS those dependent on bromodeoxyuridine, such as sister chro Chemicals. NADPH was from Boehringer Mannheim, West Germany; matid exchanges, since this nucleoside is also a substrate for 5-bromo-2'-deoxyuridine, 5-bromouracil, 5-diazouracil, 4,5<5,6)-dihydro- thymidine phosphorylase. thymine, thymine, and 5,6-dihydrothymidine were from the Sigma Chem ical Co., St. Louis, MO; 5,6-dihydrothymine was from Koch-Light Labo ratories, Colnbrook, England. Thymidine and uridine were from Schwarz INTRODUCTION Laboratories Inc., New York, NY. Ribothymidine (5-methyluridine) was from P-L Biochemicals Inc., Milwaukee, Wl. Deoxyguanosine was from The majority of laboratory tests used to monitor human pop the California Foundation for Biochemical Research, Los Angeles, CA; ulations for harmful effects of genotoxic exposures is carried out [6-3H|dThd (26.3 Ci/mmol) and |mefriy/-3H)thymine (53 Ci/mmol) were on peripheral blood leukocytes (3, 5,10). No doubt, the use of from Amersham International, Amersham, Bucks, England. Sephadex G- blood is based on the ethical considerations that such sampling 100, and protein standards for molecular weight determination (ribonu- is relatively nontraumatic and expendable to the individual. The clease, chymotrypsinogen, ovalburnin, and bovine serum albumin) were more common of these procedures such as estimations of UDS3 from Pharmacia Fine Chemicals, Uppsala, Sweden. (12), sister chromatid exchanges (14), chromosome aberrations Preparation of Platelets from PRP. Fresh PRP or outdated PRP (11, 16), and mutational frequencies (1) have all been shown to obtained from the blood bank at Lund University Hospital were prepared be dependent on the amount of exogenously supplied dThd. as described previously (13). Platelets were pelleted at 400 x g for 15 min and washed twice with 0.9% NaCI solution (saline). If isolated 1This study was supported by the Swedish Council for Planning and Coordi platelets were used, they were also cultured in physiological saline. If nation of Research in •ChemicalHealthRisks in our Environment,* by the Swedish Workers' Protection Fund, and by the Swedish National Association against Heart PRP was used, there were no additional cultural supplements present. In some cases, isolated platelets were stored as frozen pellets at -20° and Lung Diseases, and by a special grant from the Health Sciences Center in Dalby, Sweden. for up to 2 months. 2To whom requests for reprints should be addressed. Preparation of Platelet Lysates. Washed platelets were suspended 3 The abbreviations used are: UDS, unscheduled DNA synthesis; PRP, platelet- in physiological saline or in buffer solution (Na2HPO4, 5 mw, pH 7.5; or rich plasma; PPP, platelet-poor plasma; dThd, thymidine; TLC, thin-layer chroma- tography; BrdUrd, bromodeoxyuridine; BrUra, bromouracil; NA-AAF, N-acetoxy-2- ammonium carbonate, 15 mM, pH 7.2; or Tris/HCI, 50 rriM, pH 7.4). A typical preparation was carried out as follows. Platelets from 2- to 3-liter acetytarninofluorene. Received November 28,1983; accepted August 9,1984. blood were suspended in 12 ml of either physiological saline or buffer

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. R. W. Pero et al. solution, were frozen and thawed, and were sonicated on ice for 10 used for dialysis of the platelet lysate and in the subsequent determina periods of 5 sec. After another freezing and thawing, the suspension tion of dThd phosphorylase activity. was centrifuged at 20,000 x g for 30 min. In some experiments, the Measurement of Platelet Effects on UDS. Mononudear leukocytes supernatant obtained was further centrifuged at 100,000 x g for 1 hr in were isolated, treated with NA-AAF, and assayed for platelet effects on order to obtain a soluble cytosolic supernatant. UDS as described previously (13). Gel Filtration. All manipulations were carried out at 4°.Sephadex G- 100 was poured into a column (59 cm x 1.6 cm) and irrigated with 10 HIM NajHPO«plus 5 m«mercaptoethanol at pH 7.5. Standard proteins RESULTS or lysate in 0.5 ml were applied, and irrigation was continued as before. Fractions of 3 ml were collected. Protein élution was continuously Pathway of dThd Catabolism. We have reported elsewhere (12, 13) that preincubation of [3H]dThd with PRP resulted in a monitored by A2ao.The flow rate was adjusted to 0.4 ml/min. Fractions were concentrated 6- to 10-fold by freeze drying before they were time-dependent decrease in the 3H-incorporation into DNA during assayed for dThd- and thymine-catabolism. UDS. For example, a 50% reduction in the UDS value was Ion Exchange Chromatography. The standards of dThd, thymine, achieved by incubation of the precursor dThd for less than 2 hr. dihydrothymine, BrUra, and BrdUrd were chromatographed on Dowex We have also shown (12), using ion-exchange Chromatography, AG 50 W x 4 resin minus 400 mesh in columns of 24 cm x 1.1 cm and that during such preincubation, dThd was converted to thymine, at a flow rate of 0.9 ml/min according to a procedure described previously as well as to another metabolite. While this metabolite eluted (2, 9). Radioactive substrates and their products were detected by from the ion-exchange column in a region which overlapped counting the radioactivity of the fractions. Unlabeled standards were detected by reading A260values. considerably with the dThd region, it was not incorporated into TLC. Plastic sheets precoated with DEAE Cellulose MN 300 (Mach- DNA during UDS. We have used TLC carried out as described in "Materials and erey-Nagel, 5160 Doren, West Germany) were chromatographed in the Methods" to identify the dThd catabolito produced after incubat upper phase of ethylacetate:water:formic acid (60:35:5 v/v/v) as de scribed previously (4). Dihydrothymine was located by spraying with ing 1 ml of PRP with 2 /*M [3H]dThd for 5 hr at 37°.The most NaOH and p-dimethylaminobenzaldehyde as described (7). Other stand lipophilic standard, dihydrothymine, cochromatographed with the ards were located by UV light. Under these conditions, the R( values for unknown metabolite both having identical Rt values of 0.71. We the following standards were calculated: uridine, 0.11 ; dihydrothymidine, have confirmed (Chart 1/4) that dihydrothymine also cochromat 0.18; ribothymidine, 0.22; uracil, 0.37; dThd, 0.41; thymine, 0.53; and ographed with the product on the ion-exchange column. It may dihydrothymine, 0.71. be noted that (5,6)-dihydrothymine coeluted with the dThd ca- Detection of Enzyme Activity. Enzyme activities were determined by tabolite, but the major component of 4,5(5,6)-dihydrothymine did incubating samples with radioactive substrate and then separating the products by ion-exchange Chromatography or by TLC, or by a combi not (Chart 1ß). nation of both. TLC sheets were cut in 10-mm sections with the origin The conversion of thymine to dihydrothymine by PRP was at the center of one such section, and the radioactivity in each was also shown (Chart 2). This was accomplished by incubating determined. When quantification of the dThd and thymine regions was washed platelets from 2 ml of PRP at 37°for 5 hr in the presence desired, because of similar R( values there was about a 5% overlap of of 2 fiM |3Hjthymine and then analyzing the incubation mixture these regions preventing quantitative resolution of mixtures of these for dihydrothymine by TLC. Under these conditions, there was compounds in this range. Further details of incubation conditions are given in "Results." nearly 100% conversion of thymine to dihydrothymine. These data, together with the timecourse studies on dThd breakdown Role of PL Solutions and glassware free of P¡were prepared for one we have reported previously (/.e., the appearance first of thymine, set of enzyme assays designed to détermine the role of P, in dThd catabolism. Glassware was washed with concentrated HCI:95% ethanol followed by the appearance of dihydrothymine and the depletion (1:1, v/v) and then rinsed 4 times in glass-distilled water which had been of thymine) (12), and inhibition studies (see below), indicate that purified by ultrafiltration (Milli Q system, Millipore AB, Sweden). Buffer the pathway is dThd -»thymine -* dihydrothymine, as has been solution (Tris/HCI, pH 7.4, 50 HIM) prepared with ultrapure water was shown previously for other tissues (7). No further metabolism of

IHYMIDINE REGION THYMINE REGION

1O

20 30 4O 20 30 40 Elution volume (ml) Chart 1. Cochromatography of the dThd catabolite and dihydrothymine on Dowex AG 50 ion-exchange resin. PRP was incubated with 2 ¡M[3H]dThd for 5 hr at 37° and then loaded onto the resin and chromatographed as described in "Materials and Methods.' A, • •,radioactivity in the sample; • »,AMOof standard 5,6- dihydrothymine; 8, • •,radioactivity in the sample; •-•-•,A260of standard 4,5(5,6)-dihydrothymine.

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Thymine Dihydrothymine Table1 region region Contribution of platelets to the dThd catabolic activity of heparinized peripheral I i blood samples PRP was removed from 3 ml of heparinized whole blood by centrifuging at 100 i•5' PHJThymin» •—• x g for 15 min and removing the PRP. Next, the whole blood cell pellet was suspended in physiological saline to a final volume of 10 ml and then the blood [3H]Thymine »- cells pelleted again at 100 x g. The platelet-enriched saline was removed, and the NADPH saline wash step was repeated one more time. Finally, the blood cells were suspended in saline to a final volume of 3 ml, and this sample was considered to be whole blood minus PRP. A control blood sample was centrifuged under similar conditions, but the PRP was not removed nor were the blood cells washed with saline. The blood samples were incubated for 1 hr with 2 MM|JH |dThd at 37°.

dThd catabolisrrr Platelets present8 (% of thymine + (% of whole blood dihydrothymine Treatment levels) formed) OS 200 fJ of whole blood 100 37.8 R( value 2001¿of whole blood minus PRP 35.5 <5.0 Chart 2. Influence on thymine conversion to dihydrothymine of NADPH. Platelet lysate was incubated for 5 hr at 37°with either 2 MM ['HJthymine (• •),or 2 Platelet concentrations were determined by particle counting and sizing with a MM|3H|thymine and 10 mm NADPH (• •).Thevarious lysate incubations were Coulter Counter. analyzed by TLC as described in "Materials and Methods' for the presence of Quantities of dThd, thymine, and dihydrothymine were determined by TLC analysis as described in 'Materials and Methods." Less than 5% contamination of thymine and dihydrothymine. dThd with thymine or vice-versa is not detectable by this method. dihydrothymine was observed by a more detailed analysis of other Rf regions on TLC. This pathway was also confirmed by enzyme responsible has been purified from rat liver (15). It is TLC of [3H]dThd incubations with washed platelets from 3 ml of NADPH-dependent and is inhibited by diazouracil. Chart 2 shows PRP suspended in physiological saline solution or with platelet that in human platelet lysates the conversion of thymine to lysates (4 to 16 mg protein/ml) prepared in the various buffers dihydrothymine was also dependent on NADPH; we have also described above. Supernatant solutions obtained by centrifuging shown that this conversion was inhibited by diazouracil (data not such lysates at 100,000 x g for 1 hr also carried out the shown). It may be concluded that a dihydropyrimidine (dihydro aforementioned dThd catabolic pathway. thymine) dehydrogenase is the enzyme responsible. In the pres Contribution of Platelets to dThd Catabolic Activity of ence of diazouracil, dThd was converted to thymine only, indi Whole Blood. The higher values of (JDS measurements obtained cating that dihydrothymine arises from thymine as we have previously (13) when PPP was used in culture media instead of already demonstrated above. PRP suggested that most of the dThd catabolic activity was The reversibility of the thymine to dihydrothymine reaction was located in the platelets. We have therefore studied dThd catab- carried out by suspending platelets from 2 ml of PRP in 1 ml of olism in whole blood and in blood depleted of platelets. physiological saline solution fortified with 100 MM dThd. The Whole blood was cultured for 5 hr at 37°in the presence of platelets were incubated at 37°for 5 hr and then washed twice either 4 MM[3H]dThd or 100 MM[3H]dThd. After incubation, the in physiological saline. The purpose of this step was to ensure that the platelets had a supply of NADP'. A parallel incubation blood was centrifuged at 400 x g for 10 min and 2 to 3 fi\ of the PPP were subjected to TLC analysis as described in "Materials of PRP with 2 MM[3H]thymine at 37°for 5 hr was used to provide and Methods." The blood sample containing 4 MM[3H]dThd was a source of [3H]dihydrothymine. The supernatant from this in 100% converted to [3H]dihydrothymine, whereas at 100 fiu, cubation containing dihydrothymine was then used to resuspend more than 95% of dThd was converted but to [3H]thymine only. the platelets already enriched for NADP'. Next, these platelets We have also compared blood depleted of platelets with whole were incubated at 37° for 5 hr and analyzed for enzymatic activity by TLC as described in "Materials and Methods." Chart blood for dThd catabolic activity. For this purpose, 3 ml of blood were centrifuged at 100 x g for 15 min, and the PRP was 3 shows the composition of the supernatant before and after the removed. The volume of the blood cell pellet was adjusted to 10 final incubation of NADP*-enriched platelets. It is clear that the ml with physiological saline, and the sample was centrifuged platelets were provided with dihydrothymine that was converted again under the same conditions. This wash procedure was to thymine quantitatively. performed twice. The volume of the cell suspension was cor Chart 4 shows the dependence on P, of dThd conversion to rected to 3 ml with physiological saline. As a control, a sample thymine. This is consistent with the activity being dThd phospho- of whole blood was centrifuged 3 times under the same condi rylase rather than a hydrolase. The reversibility of this step in tions, but in this case no plasma was removed, and no washings whole platelets (Chart 5) was achieved by incubating 1.05 ml of were carried out. The platelet-depleted whole blood (200 ¡i\)and PRP at 37° for 5 hr in the presence of [3H]thymine (10 n\), whole blood control samples (200 /¿I)were incubated in the thymine (22 /¿I;finalconcentration, 2 ft»),deoxyguanosine (80 presence of 2 ^M pHJdThd for 1 hr at 37°.The dThd catabolic /(I; final concentration, 1 ITIM),and diazouracil (50 ¡A;final con activity was monitored by TLC analysis as we have already centration, 5 HIM). Deoxyguanosine was used to provide deox- described above. It can be seen in Table 1 that 20CM samples yribose-1-phosphate for the transferase reaction (11) catalyzed of whole blood converted 37.8% of the [3H]dThd to [3H]thymine by the phosphorylase in the reverse direction. Diazouracil was and [3H]dihydrothymine. Very littte conversion (i.e., <5%) was used to protect the thymine from conversion by the dehydrogen observed if the whole blood was depleted of platelets by replac ase. The PRP reaction mixture was subjected to TLC and radiochemical analysis as described in "Materials and Methods." ing the PRP with physiological saline. Enzymes of dThd Catabolismi. The conversion of thymine to Chart 5 shows that the major portion of thymine was converted dihydrothymine has been shown in various tissues (7, 15). The to dThd.

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invwWNt THYMNC MHY!XtOIH¥MIN£ THYHINE DIHYDROTHYMINE KGION BGION HGION THYMIDINE REGION REGION REGION ~J | |

KK3-' I 2 » —dG

HOtHICI Of 2nd INCUIAHON l U INCUIA1ION

10

os to RI Charts. Reversal of dThd phosphorylase activity in PRP (conversion of thymine to dThd). PRP was incubated for 5 hr at 37°with either 2 ¡a*[3H]thymine and 5 Charta. Reversal of the dehydrogenase reaction in PRP (conversion of dihydro thymine to thymine). [3H]dThd (2 IM) was incubated with PRP for 5 hr at 37°to rtiM diazouracil (• •),or 2 MM [3H]thymine, 5 rriM diazouracil, and 1 mm obtain a source of [3H]dihydrothymine.The product (• •)wasidentified as [3H]- deoxyguanosine (dG) (• •).TLC analyses were used to determine thymine dihydrothymine by TLC, and this was then incubated for 5 hr at 37" with platelets and dThd. isolated from PRP that had been incubated previously with 100 MMdThd (this had been done to ensure a pool of NADP*). The final incubation was also analyzed by TLC as described in •Materiateand Methods' for the production of thymine VO 670OO 25000

II

THYMWNE THYMINE MHYCWOTHYMINE REGION REGION REGION g 8«

o E P 50

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15 20 FRACTION Charte. Separation of dThd phosphorylase and dihydrothymine dehydrogenase activities present in human platelet lysate by gel filtration column chromatography. A 0.5-ml portion of platelet lysate was chromatographed on a Sephadex G-100 column as described in "Materials and Methods'; 3-ml fractions were collected, OS IßRf lyophilized, and redissolved in 0.5 ml of distilled water containing 5 mM mercapto- Chart4. Dependence of dThd phosphorylase on P,. Platelet lysate was dialyzed ethanol and 2.5 mM MgCI2; dThd phosphorylase was assayed by adding 100-/J against 10 RIM Tris-HCI, at pH 7.5, which had been prepared in ultrapure water aliquots of the column fractions containing 60 mM NasHPO< (pH 7.4), 5 mM (Milli Q system, Millipore AB). Lysate was incubated for 5 hr at 37°with either 2 mercaptoethanol, and 2.5 mM MgCI2 to 375 u\ Hanks' Eagle Medium and 25 «J io»[3HJdThdand 10 rriMNAOPH (• •),or2 MM[3HldThd, 2 mu P,. and 10 mM [3H]dThd (final concentration, 1.8 MM),and then incubating the mixture for 5 hr at NADPH (•••••);dThdcatabolismi was monitored by TLC as described in 37°.Dihydrothymine dehydrogenase was assayed by adding 10CM aliquots of the •MaterialsandMethods/ column fractions containing 60 mM Na2HPO<(pH 7.4), 5 mM mercaptoethanol, and 2.5 mM MgCl2 to 90 ¡¿ofHanks' Eagle Medium, 50 /J of NAOPH (finalconcentration, 5 mM), and 10 M!of [3Hjthymine (final concentration, 4 MM),and then incubating the Molecular Weights. Becauseof the differencesinthe molec mixture for 5 hr at 37". These assay conditions contain all the factors shown ular weights reported for dThd phosphorylase (6,9) and dihydro previouslyto be essential for both enzymatic activities (6,15). The assayed column fraction aliquots were further analyzed by TLC for either the percentage of degra thymine dehydrogenase (15), platelet lysate was subjected to dation of dThd to thymine (7/iy), dThd phosphorylase activity (• •),orthymine chromatography on gel filtration media to separate these 2 to dihydrothymine, dihydrothymine dehydrogenase activity (•••••).Protein(mg/ enzymatic activities. The data in Chart 6 reveal that we have fraction) was determined by the Lowry procedure (• •). successfully separated the 2 enzymatic activities on a Sephadex G-100 column. The phosphorylase eluted from the column rang fractions, where molecular weight estimations are not possible ing from M, 65,000 to 90,000, while the dehydrogenase was but are in excess of 100,000. These data are consistent with the excluded from the gel matrix and appeared in the void volume previously reported M, 70,000 (6) and M, 110,000 (9) for dThd

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T hym»din« Thyrnw>* Dtftydrothymtn* Table2 region f*g*oo rcgtoo Inhibition of dThd conversion to dihydrothymine by thymine II PRP samples were incubated with 2 ¡at|3H|dThd for 5 hr at 37°.Quantities of dThd, thymine, and cShydrotriymine were determined by TLC analysis as described in 'Materials and Methods.' Less than 5% contamination of dThd with thymine or vice-versa is not detectable by this method.

% of radioactivity found in the TLC regions

oil lyiiiiie73 Sample A' Samples"Sample 1894Ditiydi 76<5 C°dThd11<5<5Thymine12

* Control, no other adoWve. * Thymine (200 MM)added after 5 hr incubation period. 0 Thymine (200 «")added at the same time pHJdThd was added.

Table 3 Production of dihydroihymine in PRP after Incubation with various concentrations of thymine Radioactive |3H]thymine was mixed with unlabeted thymine to achieve |3H|- thymine concentrations of 2.5, 20.5, and 500.5 MM,respectively, after addition to 2-ml aliquots of PRP. The [3HIthymine-supptemented PRPs were then incubated for 5 hr at 37°.The conversion of thymine to dthydrothymine was determined after specific activity corrections by ion-exchange chromatography on Dowex AG 50 W x 4 as described in 'Materials and Methods.'

Thymine added to PRP (nmol) Dihydrothymineproduced(nmof) 5 5 41 8.2 1000 0

bation period of 5 hr at 37°.There was a complete conversion (i.e., 100%) of [3H]thymine to pHJdihydrothymine, regardless of the amount of unlabeled dihydrothymine that was added to the various PRP incubations. Then, 1-ml aliquots of PRP were incu bated with 2 UM [;iH|dThd (Table 2, Samples A and B) and with 2 MM[3H]dThd plus 200 UM unlabeled thymine (Table 2, Sample C). Following 5-hr incubation of all samples at 37°, thymine (unlabeled) was added to Sample B (Table 2) to a 200 /¿Mfinal concentration to provide a control for the possible effect of such levels of thymine on chromatography. After TLC analysis (Table 1.0 Rf 2), it was dear that no Chromatographie distortion had occurred and that thymine had inhibited the production of dihydrothymine. Chart7. Capacity of PRP to catabolize dThd. One-mi aliquots of PRP were incubated for 5 hr at 37° with dThd concentrations of 4 MM (• •),22 ¡a» The conversion of thymine to dihydrothymine was also checked (• •),107 MM(x x), 202 MM(O- •-O),500 MM(A A), and 1000 ¡a* by ion-exchange chromatography. As indicated previously (Chart P •);13HidThd was introduced at a standard 2-pM dose in each of the above 1X1),this technique is unsuitable for samples containing both incubations. The PRPs were analyzed for dThd catabolismi by TLC as described in •MaterialsandMethods.' dThd and dihydrothymine, but for the separation of thymine and dihydrothymine in the absence of dThd it is perfectly adequate. phosphorylase, and M, 235,000 (15) for dihydrothymine dehy- Table 3 shows the inhibitory effects of thymine on dihydrothy drogenase. mine production. These data are consistent with the dehydro- Metabolic Inhibition. The presence of dihydrothymine dehy- genase being inhibited by thymine and not by dihydrothymine. drogenase in human platelets has not been reported by other As with the dehydrogenase, it was also observed that the investigators. We have sought an explanation for the divergence dThd phosphorylase activity did not convert its substrate com of our observations from those of other workers, particularly pletely at the higher levels of 500 to 1000 UM dThd. In order to Desgranges et al. (6) who tested for, but could not detect, this test whether this was because of inhibition of the enzyme by activity. It can be seen (Chart 7) that dihydrothymine production thymine, the following experiment was carried out. One-mi ali varied with the concentration of dThd. At lower levels of dThd quots of PRP were incubated for 5 hr at 37°with 2 MM[3H]dThd (up to 202 MM),all of the substrates were converted to thymine. and varying concentrations of thymine at 0, 300, 500, 750, and Therefore, this was the level of thymine to which the dehydro- 1000 MM.In all cases, the conversion of [3H]dThd to [3H]thymine genase was exposed. In order to determine whether the enzyme was unaffected by the presence of unlabeled thymine in the PRP is inhibited by thymine or by dihydrothymine, the following ex incubations. Therefore, inhibition of the phosphorylase by thy periments were performed. One-mi aliquots of PRP were incu mine at these levels was ruled out. bated with 2 MM [3H]thymine together with dihydrothymine at Effects of Platelet Lysates on UDS. It has been clearly shown 0,15,25, 35, and 50 MM.The conversion of [3H]thymine to [3H]- that platelets influence genotoxic measurements in that they dihydrothymine was determined by TLC analysis after an incu- inhibit UDS induced by the mutagen NA-AAF (12, 13). Further

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. fí. W. Pero et al. Table4 the supernatant was analyzed by ion-exchange chromatography Effects of PRP and of various platelet lysate fractions on UDS as described in "Materials and Methods." The BrdUrd was con The procedure for estimating platelet effects on UDS is published elsewhere verted more than 90% to a compound which coeluted with BrUra. (13). Briefly, after induction of DMA damage in platelet-depleted mononuclear leukocyte cultures by a standardized 10 ¡Mdose of NA-AAF, UDS was quantified These data indicate that platelet dThd phosphorylase readily for 18 hr by pulsing with 10 ¿id/ml[3H]dThd in the presence of 10 mw hydroxyurea. catabolizes BrdUrd. The effects of the various plasmas and other additives listed below were measured by their incorporation into the cultures during the 18-hr incubation for quantifying In order to better substantiate the significance of BrdUrd UDS. catabolism for the sister chromatid exchange assay, we have of inhibition of used blood cell culture conditions similar to those used by many PlasmaaddedPRPPRP, additive0.48 UDS3596 cytogenetists (3, 5, 10). Two ml of the buffy coat from a 1% dextran T-500 sedimentation of heparinized whole blood (i.e., preincubated8 ^MpHldThd"Ratetet equal to the leukocytes and platelets from 1 ml of whole blood) PRP,preincubatedPPPPPPPPPPPPPPPOther 30047678731 were pelleted at 400 x g and resuspended in 5 ml of Hanks'

ifPlateletlysate, 50 Eagle medium containing 100 UM BrdUrd. The platelet-rich leu 100«JPlateletlysate, kocyte cultures were incubated for 24 hr at 37° and then ¡A200-MIG-1lysate, 200 00 fraction% analyzed by ion-exchange column chromatography as described 8 [3H]dThd (0.48 IM) added during 5-hr preincubation; no further [3H]dThd was above for breakdown of BrdUrd to BrUra. Again, there was more than a 90% conversion of BrdUrd to BrUra. These data were ' Following preincubation of 0.48 IM pHJdThd with PRP, further addition of pH]- dThd was made for the 18-hr period used to estimate UDS. taken as strong evidence that sister chromatid exchange deter c Standard lysate in physiological saline (see "Materials and Methods"). minations may be influenced by leukocyte cultures that contain " Lysate of platelets from 250 ml of blood. The 200 /d tested represented 50% of Fraction 5 from Sephadex G-100 chromatography as in Chart 6 and 'Materials platelets because they, in turn, catabolize BrdUrd. and Methods." DISCUSSION experiments have been performed which show the connection We have reported previously that human blood platelets inter between the enzyme activities described above and the UDS fere with UDS measurements (12,13). Preliminary observations inhibition. suggested that this resulted from dThd catabolism, and initial Human mononuclear leukocytes were exposed to NA-AAF studies on the catabolic pathway were reported (12). We have and then allowed to repair for 18 hr in the presence of a [3H]- now studied dThd catabolism by PRP, by isolated platelets, and dThd pulse as we have described previously in order to quantify by platelet lysates. UDS (13). During UDS quantification, cultures were incubated It can be concluded that the dThd catabolic activity of whole with PRP, with PRP which had been preincubated with [3H]dThd human blood is located mainly in the platelets. It must also be for 5 hr, or with PRP which had been so preincubated with [3H]- noted that BrdUrd was substantially degraded by platelets and dThd and was then fortified with an additional [3H]dThd pulse by whole blood and that was after 18- to 24-hr incubation (0.48 UM)immediately prior to UDS measurement. In another set periods. In sister chromatid exchange analyses routinely carried of experiments, PPP was used during UDS quantification to out by some investigators, incubations of whole blood cultures which was added [3H|dThd and platelet lysate, or an aliquot of are often for 48 to 72 hr (3, 5, 10). Therefore, we would like to a fraction collected by gel filtration chromatography of a platelet emphasize that account should be taken of the potential effect lysate (i.e., a fraction that corresponded to the peak of dThd platelets may have on genotoxicity tests that are dependent on phosphorylase activity eluted from the column). exogenously supplied dThd or BrdUrd. It is clear (Table 4) that the effect of dThd preincubation with While our work has again indicated the presence of dThd PRP on UDS measurements was relieved by the supply of fresh phosphorylase in human blood platelets, we have also shown [3H]dThd. It is also clear that all fractions containing dThd phos the presence of a thymine-converting enzyme. Dependence on phorylase activity lowered the level of UDS estimations. NADPH and inhibition of diazouracil indicate that the enzyme is In addition, we have permitted UDS to proceed following NA- dihydrothymine (dihydropyrimidine) dehydrogenase. We report AAF exposure without [3H]dThd but in the presence of [3H]- that this enzyme is significantly inhibited by thymine concentra thymine. No incorporation of label was detected. However, when tions in excess of 20 UM. This may be viewed in the light of a deoxyguanosine was added to cultures containing [3H]thymine previous report on the rat liver enzyme. In that case (15), the K™ for measurement of UDS, then label was incorporated because for thymine was found to be 2.5 /UM,much lower than the levels dThd phosphorylase was reversed converting [3H]thymine to at which we noted significant inhibition. We conclude that this [3H]dThd (Chart 5). Although the UDS value was only approxi inhibition is the reason that other workers (6) did not find this mately 20% of the value obtained with a comparable [3H]dThd activity in human platelets. pulse, it was a measurable UDS level (data not shown). While we have carried out preliminary substrate/product inhi Catabolism of BrdUrd. BrdUrd substitution into DNA is used bition studies, we have not made kinetic measurements with in sister chromatid exchange analysis. The reagent is used by isolated enzymes. Thus, we cannot say how the kinetic behavior some investigators at a final concentration in the test of 0.4 to of whole platelets differs from that of platelet lysates. Perhaps 10 MM (10) and by others (8) at 100 JUM.It was of interest, dihydrothymine dehydrogenase inhibition is more readily de therefore, to check the possible catabolism of the reagent in tected in lysates; this would explain (Chart 4) why thymine was whole blood, or in the presence of platelets. One ml of Hanks' not fully converted to dihydrothymine. Alternatively, since the Eagle medium fortified with BrdUrd (1.0 rriM) was incubated with lysate used in that experiment had been thoroughly dialysed, the washed platelets from the PRP isolated from 50 ml of whole result may reflect instability of the dehydrogenase, or its require blood. The platelets were then incubated for 18 hr at 37°,and ment for an ion removed by dialysis.

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In Table 1, the values for dThd catabolism are not in exact 5. Bridges, B. A., Butterworth, B. E., and Wienstein, I. B. (eds ). Indicators of Genotoxic Risk. Banbury Report 13, p. 580. Cold Spring Harbor, NY: Cold mathematical proportion to the platelet concentrations shown. Spring Harbor Laboratory, 1982. This reflects the sensitivity of platelets to the manipulations to 6. Desgranges, C., Razaka, G., Rabaud, M., and Bricaud, H. Catabolism of thymidine in human blood platelets: purification and properties of thymidine which the Wood sample had been subjected. Release of, and phosphorylase.Biochem. Biophys. Acta, 654: 211-218,1981. thus loss of, a number of factors can be expected under these 7. Fink,R. M., McGaughey,C., dine, R. E., and Fink, K. Metabolismof pyrimidine conditions. In this regard, aggregated platelets apparently do not reduction compounds in vitro. J. Biol. Chem., 276:1-8,1956. 8. Hedner, K., Hogstedt, B., Kolnig, A-M.. Mark-Vendel, E., Strombeck, B., and release the dThd catabolic enzymes, since we have shown that Mitelman,F. Relationshipbetween sister chromatid exchanges and structural human serum does not inhibit UDS, whereas human PRP from chromosome aberrations in lymphocytes of 100 individuals. Hérédités,97: the same donor does (13). On the other hand, PPP prepared 237-245,1982. from PRP conditioned for 24 hr by incubation at 37°has more 9. Heldin,OH., Wasteson, A., and Westermark, B. Partial purification and char acterization of platelet factors stimulating the multiplication of normal human dThd phosphorylase activity than does PPP prepared from fresh glial cells. Exp. Cell Res., 709:429-437,1977. PRP.4 10. Hsu, T. C. (éd.).CytogeneticAssays of Environmental Mutagens, p. 430. Totowa, NJ: Ailanheld,Osrnunand Co., 1982. 11. O'Donovan,G. A., and Heuhard,J. Pyrimidinemetabolismin microorganisms. REFERENCES Bacterio!.Rev., 34(3): 278-343,1970. 12. Pero, R. W.. Olsson,A., and Johnson, D. B. In: F. DeSerres and W. Sheridan 1. Anderson, D.. Richardson, C. R., and Davies,P. J. The genotoxic potential of (eds.), Proceedings of Conference on Genetic Consequences of Nucleotide bases and nucleosides.Mutât.Res.,97:265-272,1981. Pool Imbalance.New York: PlenumPress, in press, 1983. 2. Blattner, F. R., and Eríckson,H.P. Radid nucleotideseparationby chromatog- 13. Pero, R. W., and Vopat, C. A human platelet derived inhibitor of unscheduled raphy on ion exchange columns. Anal. Biochem., 18:220-227,1967. DNA synthesis in resting lymphocytes. Carcinogenesis(Lond.),2:1103-1110, 3. Bloom, A. D. (ed.). Guides for studies of human populations exposed to 1981. mutagenic and reproductive hazards. White Plains,NY: March of Dimes Birth 14. Perry, P. E. Induction of sister-chromatidexchanges (SCEs)by thymidine and the potentiation of mutagen-mducedSCEs in Chinese hamster ovary cells. Defects Foundation, 1981. 4. Bote, N. C. Bowen, B. W. M., Khor, K. G., and Boliska. S. A. Thin layer Mutât.Res.,709:219-229,1983. chromatography of thymidine and thymidine metabolites in enzyme assays 15. Shiotani, T., and Weber, G. Purification and properties of dihydrothymine and cell extracts. Anal. Biochem., i06: 230-237,1980. dehydrogenasefrom rat liver. J. Biol. Chem., 256: 219-224,1981. 16. Yang, S. J., Hahn, G. M., and Bagshow, M. A. Chromosome aberrations 4 Unpublished results. induced by thymidine. Exp. Ce«Res.,42:130-135,1966.

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Ronald W. Pero, Desmond Johnson and Anders Olsson

Cancer Res 1984;44:4955-4961.

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