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

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Catabolism of Exogenously Supplied Thymidine to Thymine and Dihydrothymine by Platelets in Human Peripheral Blood1 [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 NOVEMBER 1984 4955 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
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