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Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents
ChemAioi. interactions, 10 (197s) 333-338
Q ElsevierScientificPublishingCompany,Amsterdam- Printedin The Netherlands
333
INCREASED URINARY EXCRETION OF NUCLEIC ACID AND NICGTINAMIDE DERIVATIVES BY RATS AFTER TREATMENT WITH ALKYLATING AGENTS
BARBARA C. F. CHU
AND
PHILIP D. LAWLEY
Chester Beatty Research Institute, Institute of Cancer Research: Royal Cancer Hospital, Fulham Road, London S W3 6JB (Great Britain)
(ReceivedJune tSth, 1974) (RevisionteoeivedSeptember3rd, 1974)
Rats treated with di(Z-chloroethyl)methyhyamine (HN2), N-methyl-N-r&osourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine O-24 b after treatment. Ethyl methanesulphonate (EMS) and dimethylnitro~mine (DM?q) gave negative results in this respect but all five alkylating agents increase the excretion of l-methyfnicotinamide (i-meNmd). In addition, a larger quantity of 7-methylguanine (7MG)and uric acid was excreted after DMN treatment. 1,4_Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5fluorouracil (SFU), cytosine arabinoside (arac), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz[u~antI~ra~ne (?-BrMBA) gave negative results.
INTROIXJCTION
It has previously been established that rats treated with X-ray@ and MMS5 excrete significantly larger quantities of deoxyc~idine and t~ymidine in their urine &24 h after treatment. An increased amount of two nicotinamide derivatives, 6-pyr-1-meNmd and I-meNmd was also excreted after MMS treatments. This paper reports that several other alkylating agents give the same effect as MMS. However, some other carcinogens and pyrimidine base analogues gave negative results. Abbreviations:AAF, 2wetylaminofluorene;araC,cytosinearabinoside;7.BrMBA,7-bromomethyl benz[ulanthracene;dC, deoxycytidine; dichlorvos, 2,2-dichlorovinyldimethyl phosphate; DMN, dimethylnitrosamine;EMS, ethyl methanewlphonate; ENUA, Ntthyl-N-nitrosourea; ‘SFU, Sfluorouracil;HN2, di(2ch~oroetbyl)methylamine; 7MG, ‘I-methylguanine;MMS, methylmethanesulphonate; I-meNmd, l-methylni~tinamide; MNUA, ~-me~yI-~-n~tr~o~; mykran, 14 dimethan~ulphonoxybutane;opt-1.meNmd, 6-pyridoneof ~-methylni~tin~i&.
334 MATERIALS AND METHODS
Chemicals
EMS was obtained from Eustman Kodak Co., Rochester, N.Y.; DMN was obtained from Ralph N. Emmanuel Ltd., Wembley, Middx., England; MNUA, ENUA and HN2 were synthesised in this laboratory; SFU and araC were obtained from Koch-Light Laboratories, Colnbrook, Bucks., England; dichlorvos was kindly supplied by the Tunstall Laboratories, Shell Research, Sittingbourne, Kent, England; 7-BrMBA by Dr. A. Dipple, and myleran and AAF by Dr. 0. G. Fahmy, of this Institute. Methods
Female CB hooded rats, 8 weeks old, approx. 150 g, were placed in metabolic cages (Jencons Ltd., Hemel Hempstead, England) and received food (Spratt’s small laboratory animal diet) ad fibitum. HN2, DMN, EMS and araC were dissolved in 0.9% w/v sterile NaCl solution and 0.5 ml injected i.p. into each rat. With EMS at higher concentrations, 1 ml solution was injected. MNUA, ENUA and 5FU solutions were shaken in the dark for 0.5 h and 1 ml solutions injected. Myleran was injected as a suspension in 0.9% NaCl solution. 7-BrMBA, AAF and dichlorvos were dissolved in trioctanoin:EtOH (4:l) and 0.5 ml was injected. Urine was collected during the periods 24-o h before injection, O-24 h after, and 24-48 h after injection, and stored at -20” if not used immediately. After separation of urine products on Dowex AG50 x 4 (less than 400 mesh) (NHa+ form) with 0.3 M ammonium formate, pH 8.9 (as described in detail in ref. S), relevant peaks were pooled and rechromatographed on Sephadex G-10 (Pharmacia Ltd., London W5, England) with 0.05 M ammonium formate, pH 6.8. Optical density measurements were made after purification in this system by pooling appropriate chromatographic fractions and measuring their UV absorption relative to that of authentic compounds of known concentrations in the same solvents. Each peak was then again evaporated and a portion chromatographed on cellulose (Polygram 300, Camlab) TLC in two systems: System I - n-butanol saturated with water, and System II - isopropanol:conc. NHs:HsO (7: 1:2); dC, cytidine, thymidine, uracil, 6-pyr-lmeNmd and 7 MG each gave one UV spot in both systems. RESULTS
The results in Table I show that excretion of the DNA nucleosides, dC and thymidine, was significantly increased O-24 h after treatment with HN2 (5 mg/kg), MNUA (50 mg/kg) and ENUA (50 mg/kg), although with ENUA the effect at 50 mg/kg was less marked. The excretion of uracil and cytidine was also affected. With EMS (200-400 mg/kg), DMN (30 mg/kg), myleran (30 mg/kg) and dichlorvos (10 mg/kg) no significant increased excretion of these pyrimidines was detected. However, all the alkylating agents tested, except dichlorvos and myleran, increased the amount oi I-meNmd found in the urine O-24 h after treatment. The excretion of all
OF RATS BEFORE AND AFTER INJECTION OF
HN2 (5 mg/kg), MNUA (50 m&kg), ENUA (50
Uracil
Cytidine
67.0b
109.8 i 30.6 163.5 f 43.3 134.8 f 6.1
136.1+ 17.9 194.5f 37.9 143.5f 12.0
-
44.6 58.4 13.0
101.4 141.3 239.2b 268.8 JS4.7 202.8
97.8 i 11.1 150.2 i 29.7* 103.2 i 39.6
44.6 49.0 24.3
119.7 -
-
112.3 j, 23.38 -
-
233.3b 121.7 -
-
n.d. -
-
110.7i 17.6 traced 116.3& 24.3’ 183.3i 20.58 259 i 30.3s 90.0 zz 3.6
-c
Thytnidine
90.3 & 28.9 103.6 $I 39.9 71 A f 29.6
103.6& 29.2 193.1-f 32-g 105.6rt 23.2
81.8 104.4 78.1 98.6 J34.0 86.4
122.1f 38.2 116.0f 32.8 82.1 f 16.3
41.3 f 15.4 60.8 & 41.7 54.4 Tf-.13.2
7MG
IEpyr-l-
IJttpNd
1218.8 979.4
754.1
::lt
3.5
700.1 937.8 1192b
592.6 f 134.4
2.5 f 1.2 3.3 5.8 5.8
628.0 4 78.2 J 136.8 & 240.0’
3.6 f 0.3 5.4 f 0.8.
4.3 i 1.4 658.2 f 65.4 5.9 + 2.8 1164.8& 220.7” 3.0 f 1.4 493.5 &. 74.5
nJeNntd
996.3 f 269.7 1776.7 & 458.5 321.5 f 132.2
6.7 d: 0.5 2.6 f 0.5
5.1
684.8 i 109.6 1400.3 & 236.8” 827.0 & 255.9
1206.4& 309.2 5.3 zk I.2 656.b& 89.3 2276.0-+ 359.@ 6.8 i 0.5 1108.4 f 223.01 1580.7f 30.6 2.6 3 0.3 827.0 f 255.9
1535.2 1707 2319.5 2096.1 1049.55 1099.0
912.0 f 79.8 1277.3f 508.2 649.0 f 266.5
1674.3i 503.4 2198.4ziz188.2 700.7 + 310.5
Uric acid
n.d., Not determined. 5 Statisticaly significant difference from controi
DMN - 30 mg/Jcg(3) 24-Qh before 297.6 * O-24 h after 261.8 + 24-48 h after 201.5 + EMS - W mg/kg (4) 24-Oh before 189.5f O-24 h after 215.6 f 24-48 h after 142.0rt
HN2 - 5 mg/kg (3) 24-Oh before 173.7* 15.8 O-24 h after 1130 f 220.78 24-48 h after 179.9f 22.4 MNUA -50 mg/kg (3) 24-Q h before 230.9 k 39.2 O-24 h after 1070 f 28.38 24-4g h after 214.6 f 89.6 EN UA - 50 mg/kg (2) 156.9 24-Qh before 207.7 O-24 h after 496.9b 781.8 24-48 h after J43.0 167.0
dC
The amounts are given in fig excreted per rat during a 24-h period; those of 6-pyr-1-meNmd are expressed in UV absorbance units. Values refer to means and standard deviation of several determinations, the number of animals being shown in brackets. Values for ENUA eqeriments are given in duplicate.
AMOW OF UV-ABSORBWG PRODUCIS EXCRETED IN URINE mg/kgl, DMN (30 mg/kg) AM) EMS (2094oO m&kg)
TABLE I
336 these products returned to normal during the 24-48 h after treatment period. Only DMN was found to increase significantly the excretion of 7MG O-24 h after treatment, as previously reported by Craddock and Magees. Uric acid excretion was also significantly affected after DMN treatment. Myleran (50 mg/kg), dichlorvos (10 mg/kg), SFU (60 mg/kg), araC (60 mg/kg), AAF (20 mg/kg) and 7-BrMBA (30 mg/kg) gave negative results in all cases. After injection of araC (60 mg/kg), 60 “/, of the drug was recovered unchanged in the urine 48 h after treatment. DISCUSSION
The similar cytotoxic effects caused by X-rays and the radiomimetic alkylating agents on animals and proliferating cells have been frequently noted, and it has been suggested that alkylating agents and radiation exert their effects by inactivating DNA as a template for DNA replication; DNA strand breakage in the intact animal has been found for both X-rays’ and alkylating agents 8.8. The results reported here and previous1y1-5 seem to corroborate this ;iew ; X-rays and the alkylating agents MMS, HN2, MNUA and ENUA caused the excretion of significantly higher levels of dC and thymidine in the urine O-24 h after treatment. However, there are numerous excep tions to this as found in the case of EMS, DMN, myleran and dichlorvos. The positive results may either indicate those compounds which are more efficient in reacting with DNA, or those which induce similar specific DNA damage. Those compounds which gave negative results may be more easily hydrolysed and inactivated before they reach the DNA, or be too unevenly distributed in the animal to allow widespread damage to DNA. DMN and AAF must be enzymatically activated, principally in the liver, in order to react with DNA in vivd*Jl; therefore, their action is localised and this may account for the negative results obtained. EMS, however, is known to be evenly distributed throughout the organs of the rat after injectionlz. Its rate of decomposition in the rat is also considerably slower than that of ENUA, MNUA and MMSlsJs. EMS was also more efficient than ENUA in alkylating DNA and RNA of several organs of the rat 1s. Its apparent failure to yield DNA degradation products in urine cannot therefore easily be explained. In the reaction of MMS and EMS with DNA in vitro, EMS gave 2 “/, of products as Oa-ethylguanine14 and approx. I5 % as phosphotriesters 15: the corresponding values for MMS were 0.3 % (ref. 16) and 1% respectively l5. This indicates that the two compounds react differently with DNA. However ENUA gave even higher proportions of Os-ethylguanine in DNA of the rat15 and also more triesters with the RNA bacteriophage RI7 (ref. 18). Previous experiments with short-term pre-labelled DNA showed that the excreted pyrimidines after MMS treatment originated from the DNA, and not from DNA precursor materials. This was confirmed, using rats whose DNA had been stably labellcd by [14C]orotate injection during the first three weeks after birth (to be published). Excretion of the pyrimidines may result from DNA breakdown during cell necrosis, or may derive from portions of DNA excised during the repair process. Singlestrand breaks, which are then rejoined, have been shown to occur in rat liver DNA
337
after i-p. injections of various alkylating agents***, and the excreted pyrimidines may derive from this process. For those impounds which are known to react with DNA in v~~~lo~llbut which appeared to cause no significant increased excretion of the natural pyrimidines, it is possible that repair products from DNA-containing moieties derived from the carcinogens might be present in the urine, but the present methods were not designed to detect these. The antimetabolites 5FU and araC also gave negative results, although they are known to inhibit DNA synthesis in viva19**0.This indicates that inhibition of DNA synthesis per se is probably not the cause of increased urinary pyrimidine excretion. increased methylnicotinamide excretion seems to be a common effect of alkylating agents. Negative results were, however, obtained with dichlorvos, myleran, and the uralkylating agent, 7-&MBA. Although these agents are known to alkylate nucleic acids in v&u, the extents of alkylation are relatively limitedef-es_ The significance of increased nicotinamide excretion is not yet known, although it correlates well with previous reports that certain alkylating agents deplete NAD levels and inhibit glycolysis in tumour cells24.ss. Compared with the level in normal rat liver, NAD concentration was found to be lower in rapidly dividing tissues of the rat, in embryonic and newborn livers, and in livers of rats fed the carcinogen 3’methyl-4dimethylaminobenzeneas. In mice, MNUA administered intravenously reduced the NAD concentration in liver in dose-dependent fashions’. Some biochemical correlation between nicotinamide metabohsm and DNA synthesis has also been found in ~~ysuf~~ ~ly~~~~ulu~ %*.The increased excretion of nicotinamide derivatives observed here could therefore result from increased DNA synthesis as a consequence of damage by alkylating agents. Since this was not observed with X-irradiations, it also indicates that nicotinamide metabolism disturbance detectabte in this way is a specific result of alkylation damage. ACKNOWLEDGEMENTS
B. C. F. Chu wishes to thank Shell International Research fat a Research Fellowship. The work of this Institute is supported by grants from the Medical Research Councif and the Cancer Research Campaign.
REFERENCES I J. Parizek, M. Arient. Z. Dienstbierand J. Skoda. Deoxycytidinein urine as an indicator of changes after irradiation, Narwre, 182 (1958) 721-722. 2 C. D. Guri, K. F. Swingle and L. J. Cole, Urinary excretion of deoxycytidine in rats after X-irradiation: dose-responseand effect of age, intern. J. Radiatiotr Biol., 12 (1967) 355-365. 3 L. 0. Froholm, Pyrimidine excretion after irradiation in rats, Intern. J. Rudiution Biol., I2 (1967)
35-49. 4 G. B. Gerber and J. Remy-Defraigne. Mechanism of deoxycytidinuria in irradiated mice and rats, Radiation Res., 40 11969) 105-I 1I. 5 3. C. F. Chu and P. D. Hawley, Increased urinary excretion of pyrimidine and n~otinamide derivatives in rats treated with methyl ~than~uiphonate, C~~.-~~o~. fn?eruc?ion~,8 (1974) 65-73.
338 6 V. M. Craddock and P. N. Magee, Eflhct of ad~~stmtion of the carcinogen di~thylni~osamine on urinary 7.methylguanine, B&hem. J., 104 (1967) 435-440. 7 T. Gno and S. Gkada, Estimation in viva of DNA strand breaks and their rejoining inthymus and liver of mouse, Intern. J. RaaWbn Biof., 25(3) (1974) 291-301. 8 I. Damjanov, R. Cox, D. S. R. Sarma and E. Farber, Patterns of damage and repair of liver DNA induced by can5nogenic methylating agents in viw, Cancer Res., 33 (1973) 2122-2128. 9 B. W. Stewart and E. Farber, Strand breakage in rat liver DNA and its repair following administration of cyclic nitrosamines, Cancer Res., 33 (1973) 3209-3215. 10 P. N. Magee and J. M. Barnes, Carcinogenic nitroso compounds, Advan. Cancer Res., IO (1967) 163-246. 11 J. A. Miller, catcioogenesis by chemicals: an overview - G. H. A. Clowes Memorial Lecture, Cancer Z&z., 30 (1970) 559-576. 12 P. F, Swam and P. N. Magee, Niimine carcinogenesis. The alkylation of N-7 of guanine of m&ii acids of the rat by ~~hylni~~mine, ~~thyl-~-ni~o~u~ and ethyl meth~~ulphona~ B&&~TFLJ., 125 (1971) 841-847. 13 P. E Swarm, The rate of breakdown of methyl metbanesulphonate, dimethyl sulphate and Nmethyl-Ndosourea in the rat, Biochem. J., 110 (1968) 49-52. 14 P. D. Lawley, D. J. Orr and M. Jarman, Isolation and identification of products from alkylation of nucleic acids; ethyl- and isopropylpurines, Biochem. J., 145 (1975) 73-84. 15 P. Bannon and W. Verly, Alkylation of phosphates and stability of phosphate triesters in DNA, Europtran J. Biochem., 31 (1972) 103-111. 16 P. D. Lawley and S. A. Shah, Reaction of alkylating mutagens and carcinogens with nucleic acids: detection and estimation of a small extent of methylation of O-6 of guanine in DNA by methyl methanesulphonate in vitro, Chem.-Z#ioZ.Zrzteractions,5 (1972) 286-288. 17 R. Goth and M. F. Rajewsky, Persistence of OQthylguanine in rat-brain DNA: correlation with nervous system-specitJc carclnogenesis by e~y~itro~ur~, Prac. NutZ. Aead. Sri. {U.S.}, 71 (1974) 639-643. 18 K. V. Shooter, R. Howse and R. K. Merrifield, The reaction of alkylating agents with bacteriophage R17,Z3iochem. J., 137 (1974) 313-317. 19 C. Heidelberger, Fluorinated pyrimidines, Progr. Nucleic Acid Res. Mol. Biol., 4 (1965) l-50. 20 E. Frei III, 3. G. Bickers, J. S. Hewlett, M. Lane, W. V. Leary and P. W. Talley, Dose schedule and antitumor studies of arabinosylcytosine, Cancer Res., 29 (1969) 1325-1332. 21 -R. Wennerberg and G. l&froth, Formation of Fmethylguanine by dichlorvos in bacteria and micq t&m.-Biol. Znteractians, 8 (1974) 339-348. 22 P. Brookes and P. D. Iawley, In wivoreactions of isotopically-labelled alkylating agents, in L. J. Roth (Ed.), Isotopes in Experimental Pharmacology, Univ. of Chicago Press, Chicago, 1965, pp. 403414. 23 M. P. Rayman and A. Dipple, Structure and activity in chemical carcinogenesis. Comparison of the reaction of 7-BrMBA and 7-~omomethyf-l2-~th~l~nz~a]anthra~ne with mouse skin DNA Z# vivo, Biochemistry, 12 (1973) 1538-l 542. 24 H. Holzer, Action of alkylating substances on glycofysis, in P. A. Plattner (Ed.), Chemotherapy of Gmcer, Elsevier, Amsterdam, 1964, pp. 4449. 25 S. Green and A. Dobjansky, Relationship of nicotinamide adenine dinucfeotide glycohydrolase activity to adenine dinucleotide content and rate of proliferation of Ehrlich ascites turnout cells, Cutzcer Res., 27 (1967) 2261-2266. 26 L. Jedeikin, A. J. Tbomas and S. Weinhouse, Metabolism of neoplastic tissue, X. Diphosphopyridine nucleotide levels during azo dye hepatocarcinogenesis, Cancer Res., 16 (1956) 867-872. 27 P. S. Schein and S. Loftus, Streptozotocin: depression of mouse liver pyridine nucleotides, Cancer Res., 28 (1963) 1501-1506. 28 P. B. Satao and S. Shall, Control of DNA replication in Physarumpolycepha~um, Exptl. Cell Res., 69 (1971) 295-300.
Q ElsevierScientificPublishingCompany,Amsterdam- Printedin The Netherlands
333
INCREASED URINARY EXCRETION OF NUCLEIC ACID AND NICGTINAMIDE DERIVATIVES BY RATS AFTER TREATMENT WITH ALKYLATING AGENTS
BARBARA C. F. CHU
AND
PHILIP D. LAWLEY
Chester Beatty Research Institute, Institute of Cancer Research: Royal Cancer Hospital, Fulham Road, London S W3 6JB (Great Britain)
(ReceivedJune tSth, 1974) (RevisionteoeivedSeptember3rd, 1974)
Rats treated with di(Z-chloroethyl)methyhyamine (HN2), N-methyl-N-r&osourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine O-24 b after treatment. Ethyl methanesulphonate (EMS) and dimethylnitro~mine (DM?q) gave negative results in this respect but all five alkylating agents increase the excretion of l-methyfnicotinamide (i-meNmd). In addition, a larger quantity of 7-methylguanine (7MG)and uric acid was excreted after DMN treatment. 1,4_Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5fluorouracil (SFU), cytosine arabinoside (arac), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz[u~antI~ra~ne (?-BrMBA) gave negative results.
INTROIXJCTION
It has previously been established that rats treated with X-ray@ and MMS5 excrete significantly larger quantities of deoxyc~idine and t~ymidine in their urine &24 h after treatment. An increased amount of two nicotinamide derivatives, 6-pyr-1-meNmd and I-meNmd was also excreted after MMS treatments. This paper reports that several other alkylating agents give the same effect as MMS. However, some other carcinogens and pyrimidine base analogues gave negative results. Abbreviations:AAF, 2wetylaminofluorene;araC,cytosinearabinoside;7.BrMBA,7-bromomethyl benz[ulanthracene;dC, deoxycytidine; dichlorvos, 2,2-dichlorovinyldimethyl phosphate; DMN, dimethylnitrosamine;EMS, ethyl methanewlphonate; ENUA, Ntthyl-N-nitrosourea; ‘SFU, Sfluorouracil;HN2, di(2ch~oroetbyl)methylamine; 7MG, ‘I-methylguanine;MMS, methylmethanesulphonate; I-meNmd, l-methylni~tinamide; MNUA, ~-me~yI-~-n~tr~o~; mykran, 14 dimethan~ulphonoxybutane;opt-1.meNmd, 6-pyridoneof ~-methylni~tin~i&.
334 MATERIALS AND METHODS
Chemicals
EMS was obtained from Eustman Kodak Co., Rochester, N.Y.; DMN was obtained from Ralph N. Emmanuel Ltd., Wembley, Middx., England; MNUA, ENUA and HN2 were synthesised in this laboratory; SFU and araC were obtained from Koch-Light Laboratories, Colnbrook, Bucks., England; dichlorvos was kindly supplied by the Tunstall Laboratories, Shell Research, Sittingbourne, Kent, England; 7-BrMBA by Dr. A. Dipple, and myleran and AAF by Dr. 0. G. Fahmy, of this Institute. Methods
Female CB hooded rats, 8 weeks old, approx. 150 g, were placed in metabolic cages (Jencons Ltd., Hemel Hempstead, England) and received food (Spratt’s small laboratory animal diet) ad fibitum. HN2, DMN, EMS and araC were dissolved in 0.9% w/v sterile NaCl solution and 0.5 ml injected i.p. into each rat. With EMS at higher concentrations, 1 ml solution was injected. MNUA, ENUA and 5FU solutions were shaken in the dark for 0.5 h and 1 ml solutions injected. Myleran was injected as a suspension in 0.9% NaCl solution. 7-BrMBA, AAF and dichlorvos were dissolved in trioctanoin:EtOH (4:l) and 0.5 ml was injected. Urine was collected during the periods 24-o h before injection, O-24 h after, and 24-48 h after injection, and stored at -20” if not used immediately. After separation of urine products on Dowex AG50 x 4 (less than 400 mesh) (NHa+ form) with 0.3 M ammonium formate, pH 8.9 (as described in detail in ref. S), relevant peaks were pooled and rechromatographed on Sephadex G-10 (Pharmacia Ltd., London W5, England) with 0.05 M ammonium formate, pH 6.8. Optical density measurements were made after purification in this system by pooling appropriate chromatographic fractions and measuring their UV absorption relative to that of authentic compounds of known concentrations in the same solvents. Each peak was then again evaporated and a portion chromatographed on cellulose (Polygram 300, Camlab) TLC in two systems: System I - n-butanol saturated with water, and System II - isopropanol:conc. NHs:HsO (7: 1:2); dC, cytidine, thymidine, uracil, 6-pyr-lmeNmd and 7 MG each gave one UV spot in both systems. RESULTS
The results in Table I show that excretion of the DNA nucleosides, dC and thymidine, was significantly increased O-24 h after treatment with HN2 (5 mg/kg), MNUA (50 mg/kg) and ENUA (50 mg/kg), although with ENUA the effect at 50 mg/kg was less marked. The excretion of uracil and cytidine was also affected. With EMS (200-400 mg/kg), DMN (30 mg/kg), myleran (30 mg/kg) and dichlorvos (10 mg/kg) no significant increased excretion of these pyrimidines was detected. However, all the alkylating agents tested, except dichlorvos and myleran, increased the amount oi I-meNmd found in the urine O-24 h after treatment. The excretion of all
OF RATS BEFORE AND AFTER INJECTION OF
HN2 (5 mg/kg), MNUA (50 m&kg), ENUA (50
Uracil
Cytidine
67.0b
109.8 i 30.6 163.5 f 43.3 134.8 f 6.1
136.1+ 17.9 194.5f 37.9 143.5f 12.0
-
44.6 58.4 13.0
101.4 141.3 239.2b 268.8 JS4.7 202.8
97.8 i 11.1 150.2 i 29.7* 103.2 i 39.6
44.6 49.0 24.3
119.7 -
-
112.3 j, 23.38 -
-
233.3b 121.7 -
-
n.d. -
-
110.7i 17.6 traced 116.3& 24.3’ 183.3i 20.58 259 i 30.3s 90.0 zz 3.6
-c
Thytnidine
90.3 & 28.9 103.6 $I 39.9 71 A f 29.6
103.6& 29.2 193.1-f 32-g 105.6rt 23.2
81.8 104.4 78.1 98.6 J34.0 86.4
122.1f 38.2 116.0f 32.8 82.1 f 16.3
41.3 f 15.4 60.8 & 41.7 54.4 Tf-.13.2
7MG
IEpyr-l-
IJttpNd
1218.8 979.4
754.1
::lt
3.5
700.1 937.8 1192b
592.6 f 134.4
2.5 f 1.2 3.3 5.8 5.8
628.0 4 78.2 J 136.8 & 240.0’
3.6 f 0.3 5.4 f 0.8.
4.3 i 1.4 658.2 f 65.4 5.9 + 2.8 1164.8& 220.7” 3.0 f 1.4 493.5 &. 74.5
nJeNntd
996.3 f 269.7 1776.7 & 458.5 321.5 f 132.2
6.7 d: 0.5 2.6 f 0.5
5.1
684.8 i 109.6 1400.3 & 236.8” 827.0 & 255.9
1206.4& 309.2 5.3 zk I.2 656.b& 89.3 2276.0-+ 359.@ 6.8 i 0.5 1108.4 f 223.01 1580.7f 30.6 2.6 3 0.3 827.0 f 255.9
1535.2 1707 2319.5 2096.1 1049.55 1099.0
912.0 f 79.8 1277.3f 508.2 649.0 f 266.5
1674.3i 503.4 2198.4ziz188.2 700.7 + 310.5
Uric acid
n.d., Not determined. 5 Statisticaly significant difference from controi
DMN - 30 mg/Jcg(3) 24-Qh before 297.6 * O-24 h after 261.8 + 24-48 h after 201.5 + EMS - W mg/kg (4) 24-Oh before 189.5f O-24 h after 215.6 f 24-48 h after 142.0rt
HN2 - 5 mg/kg (3) 24-Oh before 173.7* 15.8 O-24 h after 1130 f 220.78 24-48 h after 179.9f 22.4 MNUA -50 mg/kg (3) 24-Q h before 230.9 k 39.2 O-24 h after 1070 f 28.38 24-4g h after 214.6 f 89.6 EN UA - 50 mg/kg (2) 156.9 24-Qh before 207.7 O-24 h after 496.9b 781.8 24-48 h after J43.0 167.0
dC
The amounts are given in fig excreted per rat during a 24-h period; those of 6-pyr-1-meNmd are expressed in UV absorbance units. Values refer to means and standard deviation of several determinations, the number of animals being shown in brackets. Values for ENUA eqeriments are given in duplicate.
AMOW OF UV-ABSORBWG PRODUCIS EXCRETED IN URINE mg/kgl, DMN (30 mg/kg) AM) EMS (2094oO m&kg)
TABLE I
336 these products returned to normal during the 24-48 h after treatment period. Only DMN was found to increase significantly the excretion of 7MG O-24 h after treatment, as previously reported by Craddock and Magees. Uric acid excretion was also significantly affected after DMN treatment. Myleran (50 mg/kg), dichlorvos (10 mg/kg), SFU (60 mg/kg), araC (60 mg/kg), AAF (20 mg/kg) and 7-BrMBA (30 mg/kg) gave negative results in all cases. After injection of araC (60 mg/kg), 60 “/, of the drug was recovered unchanged in the urine 48 h after treatment. DISCUSSION
The similar cytotoxic effects caused by X-rays and the radiomimetic alkylating agents on animals and proliferating cells have been frequently noted, and it has been suggested that alkylating agents and radiation exert their effects by inactivating DNA as a template for DNA replication; DNA strand breakage in the intact animal has been found for both X-rays’ and alkylating agents 8.8. The results reported here and previous1y1-5 seem to corroborate this ;iew ; X-rays and the alkylating agents MMS, HN2, MNUA and ENUA caused the excretion of significantly higher levels of dC and thymidine in the urine O-24 h after treatment. However, there are numerous excep tions to this as found in the case of EMS, DMN, myleran and dichlorvos. The positive results may either indicate those compounds which are more efficient in reacting with DNA, or those which induce similar specific DNA damage. Those compounds which gave negative results may be more easily hydrolysed and inactivated before they reach the DNA, or be too unevenly distributed in the animal to allow widespread damage to DNA. DMN and AAF must be enzymatically activated, principally in the liver, in order to react with DNA in vivd*Jl; therefore, their action is localised and this may account for the negative results obtained. EMS, however, is known to be evenly distributed throughout the organs of the rat after injectionlz. Its rate of decomposition in the rat is also considerably slower than that of ENUA, MNUA and MMSlsJs. EMS was also more efficient than ENUA in alkylating DNA and RNA of several organs of the rat 1s. Its apparent failure to yield DNA degradation products in urine cannot therefore easily be explained. In the reaction of MMS and EMS with DNA in vitro, EMS gave 2 “/, of products as Oa-ethylguanine14 and approx. I5 % as phosphotriesters 15: the corresponding values for MMS were 0.3 % (ref. 16) and 1% respectively l5. This indicates that the two compounds react differently with DNA. However ENUA gave even higher proportions of Os-ethylguanine in DNA of the rat15 and also more triesters with the RNA bacteriophage RI7 (ref. 18). Previous experiments with short-term pre-labelled DNA showed that the excreted pyrimidines after MMS treatment originated from the DNA, and not from DNA precursor materials. This was confirmed, using rats whose DNA had been stably labellcd by [14C]orotate injection during the first three weeks after birth (to be published). Excretion of the pyrimidines may result from DNA breakdown during cell necrosis, or may derive from portions of DNA excised during the repair process. Singlestrand breaks, which are then rejoined, have been shown to occur in rat liver DNA
337
after i-p. injections of various alkylating agents***, and the excreted pyrimidines may derive from this process. For those impounds which are known to react with DNA in v~~~lo~llbut which appeared to cause no significant increased excretion of the natural pyrimidines, it is possible that repair products from DNA-containing moieties derived from the carcinogens might be present in the urine, but the present methods were not designed to detect these. The antimetabolites 5FU and araC also gave negative results, although they are known to inhibit DNA synthesis in viva19**0.This indicates that inhibition of DNA synthesis per se is probably not the cause of increased urinary pyrimidine excretion. increased methylnicotinamide excretion seems to be a common effect of alkylating agents. Negative results were, however, obtained with dichlorvos, myleran, and the uralkylating agent, 7-&MBA. Although these agents are known to alkylate nucleic acids in v&u, the extents of alkylation are relatively limitedef-es_ The significance of increased nicotinamide excretion is not yet known, although it correlates well with previous reports that certain alkylating agents deplete NAD levels and inhibit glycolysis in tumour cells24.ss. Compared with the level in normal rat liver, NAD concentration was found to be lower in rapidly dividing tissues of the rat, in embryonic and newborn livers, and in livers of rats fed the carcinogen 3’methyl-4dimethylaminobenzeneas. In mice, MNUA administered intravenously reduced the NAD concentration in liver in dose-dependent fashions’. Some biochemical correlation between nicotinamide metabohsm and DNA synthesis has also been found in ~~ysuf~~ ~ly~~~~ulu~ %*.The increased excretion of nicotinamide derivatives observed here could therefore result from increased DNA synthesis as a consequence of damage by alkylating agents. Since this was not observed with X-irradiations, it also indicates that nicotinamide metabolism disturbance detectabte in this way is a specific result of alkylation damage. ACKNOWLEDGEMENTS
B. C. F. Chu wishes to thank Shell International Research fat a Research Fellowship. The work of this Institute is supported by grants from the Medical Research Councif and the Cancer Research Campaign.
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