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Increased concentration of 7-methylguanine and 1-methylhypoxanthine in urine of rats bearing Yoshida tumour
Cancer Letters, 9 (1980) 339-343 o Elsevier/North-Holland Scientific Publishers Ltd.
339
INCREASED CONCENTRATION OF ‘I-METHYLGUANINE AND l-METHYLHYPOXANTHINE IN URINE OF RATS BEARING YOSHIDA TUMOUR
CHRISTOPH HANSKI and GERHARD Institute
of Biology,
Research
STEHLIK*
Centre Seibersdorf,
A-2444
Seibersdorf
(Austria)
(Received 3 February 1980) (Revised version received 3 March 1980) (Accepted 13 March 1980)
SUMMARY
Sprague-Dawley rats bearing Yoshida Tumour showed an increased excretion of 7-methylguanine and 1-methylhypoxanthine with their urine as compared to control animals. The concentration of these methylated purines, which belonged to the non-specific cancer markers, was already significantly higher 2 days after tumour injection and more than 2-fold after 3 additional days. This was proved by daily L-[ “CH3]methionine supply. Separation and identification of these compounds was done by liquid chromatography and mass spectrometry. By RNA analysis it became evident that tumour RNA contained more methyl groups than liver RNA of control rats. This suggested that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to an increased turnover of tumour RNA but also to its higher degree of methylation.
INTRODUCTION
Tumour-bearing humans and animals excrete higher amounts of methylated purines than normal individuals [ 81. This has mainly been ascribed to the increased methylation found in tRNA of most tumours [ 11. The methyl group originates from S-adenosylmethionine and the methylation takes place at the macromolecular level. The S-adenosylmethionine-tRNA-methyltransferases in tumour tissues have an altered specificity and also a higher activity than those from the corresponding normal tissue [3]. The methylated purines belong to the unspecific cancer markers and their excretion has already been applied in the follow-up of tumour therapy [ 71. Recently it *To whom reprint requests should be addressed.
340
has been found that tRNA from Ehrlich ascites tumour contains 2 supernumerary methylated purines [ 51. It could be expected that Yoshida tumour growth affects the excretion of methylated purine bases in urine. In the present paper evidence is presented that the Yoshida ascites tumour of the rat causes an increased excretion of 7-methylguanine and l-methylhypoxanthine in urine. MATERIALS
AND METHODS
Five Sprague-Dawley rats (weight - 200 g each) were injected intraperitoneally with 0.3 ml Yoshida ascites fluid. Immediately afterwards animals were injected intraperitoneally with 10 VCi L-[ 14CH3 ] methionine (56 Ci/mol) in physiological saline solution. The injections of labelled methionine were repeated on the second, third and fifth day of the experiment. Controls were 3 untreated rats receiving the same methionine injections. The animals were kept in specially adapted cages and urine was collected every 5 h following each methionine injection. Approximately equal amounts of urine were collected from each rat. On the fifth day of the experiment the animals were killed and dissected. From 5 rats injected with Yoshida tumour the urine was pooled only from those 4 animals in which tumour was visible after dissection. The urine samples (vol. = 3 ml) were fractionated on Dowex 50 X 8, 400 mesh, column 1.5 X 140 cm, eluent: diluted ammonia (pH 9.8), and the 14C-radioactivity of each fraction was measured in a liquid scintillation spectrometer TRI CARB, Packard, model 3375. Mass spectra were obtained on a Varian MAT CH-5 with direct inlet system, 70 eV electron energy, 170°C ion source temperature and a resolution of m/Am = 1000 (10% valley). The mass spectrometer was equipped with a spectra system SS-100 MS. RNA was isolated and purified from 10 g liver or tumour tissue of these rats following method 2 described by Kirby [ 41. RESULTS
In the liquid chromatogram of the urine from the tumour bearing rats 2 weeks, marked A and B in Fig. 1, showed a higher radioactivity than the corresponding peaks in the chromatogram of the controls. The radioactivity of these peaks increased with the growth of the tumour (Fig. 2). The fractions of peak A and peak B were separately pooled, concentrated and rechromatographed on Sephadex G-10. The rechromatography permitted a selection of main components contributing mostly to the difference in “CH,-radioactivity between tumour and control in peaks A and B. The corresponding fractions were then dried and directly analyzed by mass spectrometry. The similarity of the obtained mass spectra with those of standards
341
10
20
30
LO Fraction
Fig. 1. graphy monia on the
50
60
70
80
90
100
number
Distribution of ‘T-radioactivity of rat urine, fractionated on a liquid chromatocolumn. Column 1.5 X 140 cm: Dowex 50 X 8, 400 mesh. Eluent: diluted am(pH 9.3) 6 ml fractions, liquid scintillation fluid prepared as in [S]., rats 5th day of Yoshida tumour growth- , **.,.....*......, control rats.
L.
3.
3.
3.
. . .L .
. /
m
m
3.
1 Days
after
2 injection
L 3 of Yoshlda-turnour
5
Fig. 2. Excretion of methylated compounds with the urine of tumour bearing (0 and 0) and control (o and n) rats. The diagram represents the change of *%H,-radioactivity of peak A (e and 0) and peak B (c and 0) from the liquid chromatogram exemplified by Fig. 1.
342
strongly indicated 7-methylguanine and 1-methylhypoxanthine as components of peak A and B, respectively (Figs. 3 and 4). The UV-spectra of the fractions belonging to peak A confirmed that its main component was 7-methylguanine. The incorporation of the 14CH,-group of L- [ 14CH,]methionine into liver RNA was very low (430 + 30 cpm/mg) as compared to tumour RNA (21,920 + 1300 cpm/mg). DISCUSSION
The excretion of increased amounts of 7-methylguanine in urine of tumour bearing mammals has already been reported [ 11. l-hlethylhypoxanthine probably derives from l-methylinosine which has also been found in higher amounts in the urine of cancer patients [ 21.
u
150
0 160
m/e
50
100
150
m/e
200
260
165
96
0
50
r.
x)0
150m,e
LY”
m/e
Fig. 3. (a) Mass spectrum of the component of peak A which. mostly contributed to the difference in radioactivity between tumour and control. (b) Mass spectrum of 7-methylguanine. Fig. 4. (a) Mass spectrum of the component of peak B which mostly contributed to the difference in radioactivity between tumour and control. (b) Mass spectrum of l-methylhypoxanthine.
343
After metabolic degradation of tRNA the methylated bases do not undergo demethylation nor are they incorporated into nucleic acids but are excreted as bases or as nucleosides with the ribose moiety. Therefore the excretion of these bases can be related to changes in methylation and/or turnover of the tRNA. After RNA-extraction and radioactivity measurement it was evident that tumour RNA contained more methyl groups than the RNA from the liver of control rats. This suggests that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to the increased turnover of tumour ribonucleic acids but also to its higher degree of methylation. REFERENCES Borek, E. and Kerr, S.J. (1972) Atypical transfer RNAs and their origin in neoplastic cells. Adv. Cancer Res., 15, 163-190. Chang, S.Y., Lakings, D.B., Zumwait, R.W., Gehrke, C.W. and Waalkes, T.P. (1974) Quantitative determination of methylated nucleosides and pseudouridine in urine by gas-liquid chromatography. J. Lab. Clin. Med., 83 816-830. Kerr, S.J. and Borek, E. (1972) The t-RNA methyltransferases. Adv. Enzymol., 35, l-27. Kirby, K.S. (1965) Isolation and characterization of ribosomal ribonucleic acid. Biochem. J., 96, 266-269. Kuchino, Y. and Borek, E. (1978) Tumour-specific phenylalanine t-RNA contains two supernumerary methylated bases. Nature, 271, 126-129. Lawley, P.D. and Shah, S.A. (1972) Methylation of RNA by the carcinogens DMS, MNUA and MNNG. Comparisons of chemical analyses at the nucleoside and base levels. Biochem. J., 128, 117-132. WaaIkes, T.P. and Borek, E. (1976) The metabolism of t-RNA in tumour tissue: an approach to diagnosis. Rend. Atti Accad. Sci. Med. Chir., 130, 321-333. Weissmann, B., Bromberg, P.A. and Gutman, A.B. (1957) The purine bases of human urine. I. Separation and identification. J. Biol. Chem., 224,407422.
339
INCREASED CONCENTRATION OF ‘I-METHYLGUANINE AND l-METHYLHYPOXANTHINE IN URINE OF RATS BEARING YOSHIDA TUMOUR
CHRISTOPH HANSKI and GERHARD Institute
of Biology,
Research
STEHLIK*
Centre Seibersdorf,
A-2444
Seibersdorf
(Austria)
(Received 3 February 1980) (Revised version received 3 March 1980) (Accepted 13 March 1980)
SUMMARY
Sprague-Dawley rats bearing Yoshida Tumour showed an increased excretion of 7-methylguanine and 1-methylhypoxanthine with their urine as compared to control animals. The concentration of these methylated purines, which belonged to the non-specific cancer markers, was already significantly higher 2 days after tumour injection and more than 2-fold after 3 additional days. This was proved by daily L-[ “CH3]methionine supply. Separation and identification of these compounds was done by liquid chromatography and mass spectrometry. By RNA analysis it became evident that tumour RNA contained more methyl groups than liver RNA of control rats. This suggested that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to an increased turnover of tumour RNA but also to its higher degree of methylation.
INTRODUCTION
Tumour-bearing humans and animals excrete higher amounts of methylated purines than normal individuals [ 81. This has mainly been ascribed to the increased methylation found in tRNA of most tumours [ 11. The methyl group originates from S-adenosylmethionine and the methylation takes place at the macromolecular level. The S-adenosylmethionine-tRNA-methyltransferases in tumour tissues have an altered specificity and also a higher activity than those from the corresponding normal tissue [3]. The methylated purines belong to the unspecific cancer markers and their excretion has already been applied in the follow-up of tumour therapy [ 71. Recently it *To whom reprint requests should be addressed.
340
has been found that tRNA from Ehrlich ascites tumour contains 2 supernumerary methylated purines [ 51. It could be expected that Yoshida tumour growth affects the excretion of methylated purine bases in urine. In the present paper evidence is presented that the Yoshida ascites tumour of the rat causes an increased excretion of 7-methylguanine and l-methylhypoxanthine in urine. MATERIALS
AND METHODS
Five Sprague-Dawley rats (weight - 200 g each) were injected intraperitoneally with 0.3 ml Yoshida ascites fluid. Immediately afterwards animals were injected intraperitoneally with 10 VCi L-[ 14CH3 ] methionine (56 Ci/mol) in physiological saline solution. The injections of labelled methionine were repeated on the second, third and fifth day of the experiment. Controls were 3 untreated rats receiving the same methionine injections. The animals were kept in specially adapted cages and urine was collected every 5 h following each methionine injection. Approximately equal amounts of urine were collected from each rat. On the fifth day of the experiment the animals were killed and dissected. From 5 rats injected with Yoshida tumour the urine was pooled only from those 4 animals in which tumour was visible after dissection. The urine samples (vol. = 3 ml) were fractionated on Dowex 50 X 8, 400 mesh, column 1.5 X 140 cm, eluent: diluted ammonia (pH 9.8), and the 14C-radioactivity of each fraction was measured in a liquid scintillation spectrometer TRI CARB, Packard, model 3375. Mass spectra were obtained on a Varian MAT CH-5 with direct inlet system, 70 eV electron energy, 170°C ion source temperature and a resolution of m/Am = 1000 (10% valley). The mass spectrometer was equipped with a spectra system SS-100 MS. RNA was isolated and purified from 10 g liver or tumour tissue of these rats following method 2 described by Kirby [ 41. RESULTS
In the liquid chromatogram of the urine from the tumour bearing rats 2 weeks, marked A and B in Fig. 1, showed a higher radioactivity than the corresponding peaks in the chromatogram of the controls. The radioactivity of these peaks increased with the growth of the tumour (Fig. 2). The fractions of peak A and peak B were separately pooled, concentrated and rechromatographed on Sephadex G-10. The rechromatography permitted a selection of main components contributing mostly to the difference in “CH,-radioactivity between tumour and control in peaks A and B. The corresponding fractions were then dried and directly analyzed by mass spectrometry. The similarity of the obtained mass spectra with those of standards
341
10
20
30
LO Fraction
Fig. 1. graphy monia on the
50
60
70
80
90
100
number
Distribution of ‘T-radioactivity of rat urine, fractionated on a liquid chromatocolumn. Column 1.5 X 140 cm: Dowex 50 X 8, 400 mesh. Eluent: diluted am(pH 9.3) 6 ml fractions, liquid scintillation fluid prepared as in [S]., rats 5th day of Yoshida tumour growth- , **.,.....*......, control rats.
L.
3.
3.
3.
. . .L .
. /
m
m
3.
1 Days
after
2 injection
L 3 of Yoshlda-turnour
5
Fig. 2. Excretion of methylated compounds with the urine of tumour bearing (0 and 0) and control (o and n) rats. The diagram represents the change of *%H,-radioactivity of peak A (e and 0) and peak B (c and 0) from the liquid chromatogram exemplified by Fig. 1.
342
strongly indicated 7-methylguanine and 1-methylhypoxanthine as components of peak A and B, respectively (Figs. 3 and 4). The UV-spectra of the fractions belonging to peak A confirmed that its main component was 7-methylguanine. The incorporation of the 14CH,-group of L- [ 14CH,]methionine into liver RNA was very low (430 + 30 cpm/mg) as compared to tumour RNA (21,920 + 1300 cpm/mg). DISCUSSION
The excretion of increased amounts of 7-methylguanine in urine of tumour bearing mammals has already been reported [ 11. l-hlethylhypoxanthine probably derives from l-methylinosine which has also been found in higher amounts in the urine of cancer patients [ 21.
u
150
0 160
m/e
50
100
150
m/e
200
260
165
96
0
50
r.
x)0
150m,e
LY”
m/e
Fig. 3. (a) Mass spectrum of the component of peak A which. mostly contributed to the difference in radioactivity between tumour and control. (b) Mass spectrum of 7-methylguanine. Fig. 4. (a) Mass spectrum of the component of peak B which mostly contributed to the difference in radioactivity between tumour and control. (b) Mass spectrum of l-methylhypoxanthine.
343
After metabolic degradation of tRNA the methylated bases do not undergo demethylation nor are they incorporated into nucleic acids but are excreted as bases or as nucleosides with the ribose moiety. Therefore the excretion of these bases can be related to changes in methylation and/or turnover of the tRNA. After RNA-extraction and radioactivity measurement it was evident that tumour RNA contained more methyl groups than the RNA from the liver of control rats. This suggests that the increased excretion of 7-methylguanine and 1-methylhypoxanthine should not be only ascribed to the increased turnover of tumour ribonucleic acids but also to its higher degree of methylation. REFERENCES Borek, E. and Kerr, S.J. (1972) Atypical transfer RNAs and their origin in neoplastic cells. Adv. Cancer Res., 15, 163-190. Chang, S.Y., Lakings, D.B., Zumwait, R.W., Gehrke, C.W. and Waalkes, T.P. (1974) Quantitative determination of methylated nucleosides and pseudouridine in urine by gas-liquid chromatography. J. Lab. Clin. Med., 83 816-830. Kerr, S.J. and Borek, E. (1972) The t-RNA methyltransferases. Adv. Enzymol., 35, l-27. Kirby, K.S. (1965) Isolation and characterization of ribosomal ribonucleic acid. Biochem. J., 96, 266-269. Kuchino, Y. and Borek, E. (1978) Tumour-specific phenylalanine t-RNA contains two supernumerary methylated bases. Nature, 271, 126-129. Lawley, P.D. and Shah, S.A. (1972) Methylation of RNA by the carcinogens DMS, MNUA and MNNG. Comparisons of chemical analyses at the nucleoside and base levels. Biochem. J., 128, 117-132. WaaIkes, T.P. and Borek, E. (1976) The metabolism of t-RNA in tumour tissue: an approach to diagnosis. Rend. Atti Accad. Sci. Med. Chir., 130, 321-333. Weissmann, B., Bromberg, P.A. and Gutman, A.B. (1957) The purine bases of human urine. I. Separation and identification. J. Biol. Chem., 224,407422.