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Influence of azacytidine on the induction of tyrosine aminotransferase and the NAD metabolism in rat liver
0020-711X/84$3.00+ 0.00 Copyright Q 1984Pergamon Press Ltd
1~. J. Bioch~m.Vol. 16, No. 12, Pp. 1387-1390.1984 Printed in Great Britain. All rights reserved
INFLUENCE OF AZACYTIDINE ON THE INDUCTION OF TYROSINE AMINOTRANSFERASE AND THE NAD METABOLISM IN RAT LIVER* H. KRUGER, R. GR,%TZand H. GRAHN Robert Koch-Institut,
Nordufer 20, D-1000 Berlin 65, F.R.G. [Tel. (030) 45-032301 (Received 22 November 1983)
5-Azacytidine was found to inhibit the induction of tyrosine aminotransferase caused by L-tyrosine and hydrocortisone. The inhibitory effect can be overcome by L-methionine. Abstract-l.
2. There was only a slight inhibition of the tryptophan induction by Sazacytidine in normal animals 4 hr after the administration of tryptophan. At 8 and 12 hr, a superinduction could be observed. 3. The NAD content in adrenale~tomiz~ animals increased after application of tryptophan and Sazacytidine. 4. There was a slight inhibition of ADPR transferase in the rat liver.
INTRORUCTION
RESULTS
Azacytidine is known to have a number of effects such as the inhibition of the synthesis of protein, RNA and DNA (CihBk, 1974). It is characterized by its bacteriostatic, carcinostatic, mutagenic, immunosuppressive and virostatic action. Azacytidine is also capable of inducing differentiation (Constantinides et al., 1977; Taylor and Jones, 1979). This has been associated with an interference with the methylation of DNA (Olson, 1979; Jones and Taylor, 1980). Furthermore, 5-azacytidine influences the induction of the enzyme, tyrosine aminotransferase 198 1). Tyrosine amino(Cihik and Kriiger, transferase may be induced by its substrate as well as by cortisone and tryptophan (see Kriiger et al., 1968). We supposed from the effect of tryptophan that the NAD adenoribosylation metabolism is of importance in the induction of tyrosine aminotransferase (Kriiger and G&z, 1980). In the present paper we report on the influence of 5-azacytidine on the induction of tyrosine aminotransferase and on the NAD adenoribosylation metabolism.
(1) Influence of 5-azacytidine in the induction of
METHODS (1) Determ~not~o~ of tyrosine ami~otra~~eruse. As reported earlier (Kriigeer and Greuer. 19651. 12) Determinkon- of NAD. The &al content of NAD + NADH, was determined according to Nisselbaum and Green (1969). (3) Isolation ofnuclei. This was performed as described by Blobel and Potter (1966). (4) Determj~uiion of ADPR tronsferuse. Kidwell and Burdette’s (1974) method was used.
*This paper is dedicated to Professor Dr Georg Henneberg on the occasion of his 75th birthday. Abbreviations: ADPR-adenosinediphosphate~bose; AZ5-azacytidine; NAD-nicotinamideadenindinucleotide; TAT--tyrosine aminotransferase.
tyrosine aminotransferase
After application of L-tyrosine the activity of the TAT increased by c. 10 times (Table 1). This effect was reduced to a large extent by .5-azacytidine. Simultaneous administration of L-methionine overcame the inhibition caused by 5-azacytidine. L-Methionine on the other hand did not influence the L-tyrosine induction. Hydrocortisone brought about a tenfold rise of the activity of the TAT (Table 2). 5-Azacytidine caused decreased which could be completely overcome by L-methionine. TAT can be induced by tryptophan, too (Kriiger and G&z, 1980; Kriiger et al., 1968). There was only a slight inhibition of the tryptophan induction of TAT by 5-azacytidine (Table 3). This could be reversed by L-methionine. We have analyzed the time course of the azacytidine effect as well. From Table 4 it can be seen that 8 and 12 hr after AZ administration a superinduction was observed. As in a previous study (Kriiger and Greuer, 1966), there was a marked potentiation of the steroid effect on the induction of TAT by L-tyrosine. This can also be seen in Table 5. 5-Azacytidine inhibited this effect. In the presence of L-methionine, the inhibition was almost non-existent. of 5-azacytidine Injuence adenoribosylation metabolism
(2)
on
the
NAD
From earlier results we supposed that the NAD adenoribosylation metabolism was involved in the process of TAT induction (Kriiger and G&z, 1978, 1980). Therefore we studied if the NAD synthesis and the activity of the ADPR synthetase were influenced by 5-azacytidine. In normal rats, there was an inhibition of the NAD synthesis caused by DL-tryptophan in the liver (Table 6). There was no effect on the NAD synthesis initiated by nicotinamide. In adrenalectomized rats, however, rhe
1387
H.
1388 Table
KRUGER et al.
I. Influence of S-azxytidine on the mduction of tvrosine aminotransferase caused by L-tyrosine in normal an&Is Treatment
pm01 p-hydroxyphenylpyruvic acid!mg protein/hr
n
L-tyrosine” (300 mg/kg)
5
3.38 i 0.93
L-tyrosine (300 mgjkg) 5-aracytldine (25 mg/kg)
4
I .08 f 0.27
L-tyrosme (300 q/kg) L-methionine (300 mg:kg)
4
3.09 * I .53
4
3.24 k 0.52
L-tyrosine (300 mg/kg) L-methmnine (300 mg/kg) 5-azacytidme (25 &kg) 5-azacytidine (25 mgikg)
4
0.85 ? 0.14
0.15MNaCl
4
0.35 * 0.03
(IOmljkg)
“Treatment over a period of 4 hr.
Table 2. Influence of 5.azacytidine on the induction of tyrosine aminotransferase caused bv hvdrocortisone in adrenalectomized animals pm01 p-hydroxyphenylpyruvic acid/mg protein/hr
Treatment
n
Hydrocortisone” (IO mg/kg)
4
3.93 f I .oo
Hydrocortisone (10 mg/kg) 5-azacytidine (25 mg/kg)
5
1.77 k 0.36
Hydrocortisone (IO mg/kg) 5-azacytidine (25 m/kg) L-methionine (300 mgikg)
5
3.80 + 1.02
5-azacytidine (25 mgjkg)
3
0.30 f 0.08
0. I5 M NaCl (10 ml/kg)
4
0.43 i 0.07
“Treatment over a period of 4 hr.
Table 3. Influence of 5-ardcytidine on the induction of tyrosine aminotransferase by tryptophan firno p-hydroxyphenylpyruwc acid/mg proteinjhr
n
Treatment oL-tryptophan” (300 mgjkg)
3
3.43 * 0. I9
tx-tryptophan (300 m&kg) 5-azacytidine (25 mgjkg)
4
2.57 * 0.88
oL-tryptophan (300 mgikg) 5.azacytidine (25 mg/kg) + L-methionine (300 mglkg)
4
3.49 * 0.99
0.15 M NaCl (I 0 ml:ke)
3
1.33 + 0.26
“Termination of the experiments after 4 hr
Table 4. Influence of 5.azacytidine on the induction of tyrosine aminotransferase 4hr
8 hr
l2hr
NaCl (0.15 M: IO ml/kg)
1.41” * 1.41 (4)
I .30 f 0.35 (5)
0.84 k 0.09 (4)
mtryptophan
2.75 i 2.00 (5)
7.07 * 0.34 (4)
1.95 * 0.55 (5)
1.55 i 0.51 (5)
3.31 f0.13 (5)
3.96 i I .48 (5)
Treatment
(500 mglkg)
5-azacytidine (25 mg/kg) wtryptophan (500 mg/kg) + S-azacytidine (25 mg/kg)
3.08f0.17 8.65 + 2.32 (5) (5) The substances were applied intraperitoneally at times zero. “pm01 p-hydroxyphenylpyruvic acid/mg protein/hr.
NAD content increased if tryptophan was applied together with Sazacytidine (Table 7). We have also tested the influence on the activity of ADPR transferase. From Table 8 it can be seen that azacytidine inhibited ADPR transferase to some extent.
5.98 f I .94 (6)
DISCUSSION
Although there are many mechanisms by which gene expression can be controlled, the role of gene methylation is receiving wide attention. In some cases a methylated gene is inactive, while hypomethylation
1389
Azacytidine on amino transferase and NAD metabolism Table 5. Influenceof 5-azacytidine
on the induction of tyrosine aminotran5fe~ds~ substances in adrenalectomized rats
by various Treatment ._~ 5-azacytidine (25 mg/kg) r-tyrosine
pmol p-hydroxyphenyipyruvic acid/q protein/hr
__-__n
(300 mgjkg)
4
0.37 ?r 0.01
5
0.46 ? 0.16
(2.5 mg/kg)
5
I .88i: 0.65
I.-tyrosine (300mg/kg) Hydrocortisone (2.5 mgjkg)
4
4.85 _+0.88
4
I .4s
4
3.66 i: 2.35
5
0.5oco.12
Hydrocorrisone
L-tyrosine (300mg/kg) Hydrocortisone (2.5 mgjkg) S-aracytidine (25 m&kg) L-tyrosine (300 mg/kg) Hydrocortisone (2.5 mg/kg) S-azacytidine (25 mgikg) t.-methionine (300 mg/kg) 0.15 M Nafl” “Treatment
(10 ml/kg)
i 0.3
I
over a period of 4 hr
Table 6. NAD synthesis
in difl’erent organs of normal Liver
-.O.lSMNaCI”
(IOmljkg)
O.lSMNdCl 5-azacytidine
(lOml/kg) (25 mg/kg)
rats under the influence of 5-azacytidine Brain
Spleen
Kidney
0.33 1 0.05 (8)
0.29 i 0.03 (7)
0.67 k 0.03 (7)
0.78~0.1s (4)
0.28 i_ 0.02 (S)
0.34 * 0.05 (5)
0.75 * 0.13 (S)
._____ 0.81” & 0.07 (6)
Nicotinamide
(500 mg/kg)
2.57 f 0.35 (4)
0.42 + 0.06 (4)
0.51 + 0.05 (4)
I .2s
Nicotinamide S-azacytidine
(SO0 mg’kg) (25 mg/kg)
2.53 f 0.30 (4)
0.29 + 0.09 (5)
0.47 i 0.06 (5)
1.41 -f-0.33 (S)
(300 mg/kg)
1.39*0.10 (4)
0.30 + 0.01 (4)
0.31 i 0.08 (4)
0.90 IO.08 (4)
oL-tryptophan (300 mg/kg) S-azacytidine (25 mg:kg)
1.00~0.17 (5)
0.30 * 0.07 (5)
0.26 f 0.03 (S)
0.76 -f 0.12 (5)
DL-tryptophan
~e~ination of the experiments ‘flrnol,ig wt NAD + NADH,.
Table
7. NAD
after 4 hr.
synthesis
in different organs of adrenalectomized influence of S-atacytidine Liver
Spleen
rats under
the
Kidney
Nicotinamide
(SO0 mgikg)
2.51” IO.64 (3)
0.37 * 0.04 (4)
1.13io.11 (4)
rx-tryptophan
(300 n&kg)
o.E%*o.13 (3)
0.23 + 0.03 (4)
0.59 + 0.0s (4)
O.R6i: 0.20 (4)
0.32 IO.06 (4)
0.72 + 0.06 (4)
S-azacytidine
+ 0.05 (3)
(25 m&g)
5-azacytidine Nicotinamide
(25 mg/kg) (SO0 mg/kg)
2.43 + 0.16 (4)
0.42 i_ 0.05 (4)
I .44 +
S-azacytidine oL-tryptophan
(25 mgikg) (300 mg/kg)
l.l6&0.09 (4)
0.33 * o.oi (4)
0.87 & 0.05 (4)
0.15 M N&l*
(~Omi~kg)
0.7 I * 0.09 101
0.25 z 0.04 10%
0.64 jlo.19 (91
“pmol/g wt NAD + NADH,. hTermination of the experiments
0.25
(4)
after 4 hr.
is necessary for gene expression (Felsenfeld and McGhee, 1982). According to this hypothesis, the presence of AZ in the DNA would block the methylation and concomitant inactivation of the regulatory in the continued inhibition of genes, resulting mammalian differentiation (Bolander, 1983). Recent reports suggest that it may not be suf%cient (Wilk et at., 1982; Ott et ai., 1982). In this paper, it is demonstrated that the inhibition of the enzyme induction of tyrosine aminotransferase by 5-azacytidine does not take place in the presence
of methionine. Whether this is due to interference with the DNA methylation has to be further elucidated. On the other hand, an influence of AZ on the NAD synthesis provoked by the injection of tryptophan was seen. Interestingly by that the NAD content increased in the liver of adrenalectomized animals if both tryptophan and AZ were present. This could have been due to an interference with ADPR transferase because the same phenomenon was found with inhibitors of this enzyme (Kriiger and in preparation) and with G&z, manuscript
H. KRUGER et al.
1390 Table 8. Influence of S-azacytidine, m-tryptophan and nicotinamide on the activity of the ADPR transferase in liver nuclei from normal rats Treatme&
cpm/mg DNAh
oL-tryptophan (500 mg/kg) 5.azacytidine (25 mgjkg)
4117
oL-tryptophan
4161
(500 mg/kg)
JonesP.A. and Taylor S. M. (1980) Cellular differentiation,
Nicotinamide (500 mg:kg) 5-azacytidine (25 mg/kg)
4203
Nicotinamide (500 mg/kg)
4.455
5-azacytidine (25 mg/kg)
4128
0.15 M NaCl (lOml/kg)
4650
“Termination 2 hr after application. “Incubation with labelled NAD for 10 min.
D-galactosamine (Kriiger et al., 1983). Further are required to elucidate this point.
studies
REFERENCES Blobel G. and Potter V. R. (1966) Nuclei from rat liver: Isolation method that combines with high yield. Science 154, 1662-1665. Bolander F. F. Jr (1983) The effect of 5-azacytidine on mammary gland differentiation in vitro. Biochem. biophys. Res. Commun. 111, 150-155. Cihak A. (1974) Biological effects of 5-azacytidine in eukaryotes. Oncology 30, 405-422. Cihik A. and Kriiger H. (1981) Tryptophan-mediated induction of hepatic enzymes stimulated by 5-azacytidine pretreatment. Biomedicine 34, 18-22. Constantinides P. G., Jones P. A. and Gevers W. (1977) Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature 267, 364-366. Felsenfeld G. and McGhee J. (1982) Methylation and gene control. Nature 296, 602-93.
cytidine analogs and DNA methylation. Cell 20, 85-93. Kidwell W. R. and Burdette K. E. (1974) Poly(ADPribose)synthesis and cell division. b’iochem. hiophys. Res. Commun. 61, 766773. Kriiger H. and Greuer B. (1965) Einflul3 von Antagonisten des Nucleinsaure-Stoffwechsels und von R(intgenstrahlen auf die Induktion von Enzymen. Biochem. Z. 341, 190-198. KrBger H. and Greuer B. (1966) Influence of cortisone on the substrate-induction of enzymes in rat liver. Nature 210, 200-201. Kriiger H., Lawel M. and Kessel H. (1968) Induktion der Tyrosin-Transaminase in Gegenwart von L-Tryptophan. Hoppe-Seyler’s Z. physiol. Chem. 349, 1221-1224. Kriiger H. and Gritz R. (1978) Induktion der TyrosinTransaminase (EC 2.1.5.1) in der Rattenleber durch NicotinsCureamid. J. clin. Chem. clin. Biochem. 16, 525-529. Kr6ger H. and G&z R. (1980) On the induction of tyrosine aminotransferase (TAT) by tryptophan and methionine. Int. J. Biochem. 12, 575-582. Krager H., Gr%tz R. and Grahn H. (1983) Influence of D-galactosamine upon the NAD-metabolism in rat liver. Inf. J. Biochem. 15, 1131-1136. Nisselbaum J. S. and Green S. (1969) A simple ultramicro method for determination of pyridine nucleotides in tissues. Analyt. Biochem. 27, 2 12-2 17. Olson C. B. (1979) 5-Methylcytosine. 5-azacytidine and development: A synthesis. Speculation Sci. Technol. 2, 365-373. Ott M.-O., Sperling L., Cassio D., Levilliers J., Sala-Trepat J. and Weiss M. C. (1982) Undermethylation at the 5-end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture. Cell 30, 825-833. Taylor S. M. and Jones P. A. (1979) Multiple new phenotypes induced in 10T; and 3T3 = cells treated with 5-azacytidine. Cell 17, 771-719. Wilk A. F., Cozens P. J., Mattag J. W. and Jost J. P. (1982) Estrogen induces a demethylation at the 5’ end of the chicken vitellogenin gene. Proc. natn Acad. Sci. U.S.A. 79, 4252-4255.
1~. J. Bioch~m.Vol. 16, No. 12, Pp. 1387-1390.1984 Printed in Great Britain. All rights reserved
INFLUENCE OF AZACYTIDINE ON THE INDUCTION OF TYROSINE AMINOTRANSFERASE AND THE NAD METABOLISM IN RAT LIVER* H. KRUGER, R. GR,%TZand H. GRAHN Robert Koch-Institut,
Nordufer 20, D-1000 Berlin 65, F.R.G. [Tel. (030) 45-032301 (Received 22 November 1983)
5-Azacytidine was found to inhibit the induction of tyrosine aminotransferase caused by L-tyrosine and hydrocortisone. The inhibitory effect can be overcome by L-methionine. Abstract-l.
2. There was only a slight inhibition of the tryptophan induction by Sazacytidine in normal animals 4 hr after the administration of tryptophan. At 8 and 12 hr, a superinduction could be observed. 3. The NAD content in adrenale~tomiz~ animals increased after application of tryptophan and Sazacytidine. 4. There was a slight inhibition of ADPR transferase in the rat liver.
INTRORUCTION
RESULTS
Azacytidine is known to have a number of effects such as the inhibition of the synthesis of protein, RNA and DNA (CihBk, 1974). It is characterized by its bacteriostatic, carcinostatic, mutagenic, immunosuppressive and virostatic action. Azacytidine is also capable of inducing differentiation (Constantinides et al., 1977; Taylor and Jones, 1979). This has been associated with an interference with the methylation of DNA (Olson, 1979; Jones and Taylor, 1980). Furthermore, 5-azacytidine influences the induction of the enzyme, tyrosine aminotransferase 198 1). Tyrosine amino(Cihik and Kriiger, transferase may be induced by its substrate as well as by cortisone and tryptophan (see Kriiger et al., 1968). We supposed from the effect of tryptophan that the NAD adenoribosylation metabolism is of importance in the induction of tyrosine aminotransferase (Kriiger and G&z, 1980). In the present paper we report on the influence of 5-azacytidine on the induction of tyrosine aminotransferase and on the NAD adenoribosylation metabolism.
(1) Influence of 5-azacytidine in the induction of
METHODS (1) Determ~not~o~ of tyrosine ami~otra~~eruse. As reported earlier (Kriigeer and Greuer. 19651. 12) Determinkon- of NAD. The &al content of NAD + NADH, was determined according to Nisselbaum and Green (1969). (3) Isolation ofnuclei. This was performed as described by Blobel and Potter (1966). (4) Determj~uiion of ADPR tronsferuse. Kidwell and Burdette’s (1974) method was used.
*This paper is dedicated to Professor Dr Georg Henneberg on the occasion of his 75th birthday. Abbreviations: ADPR-adenosinediphosphate~bose; AZ5-azacytidine; NAD-nicotinamideadenindinucleotide; TAT--tyrosine aminotransferase.
tyrosine aminotransferase
After application of L-tyrosine the activity of the TAT increased by c. 10 times (Table 1). This effect was reduced to a large extent by .5-azacytidine. Simultaneous administration of L-methionine overcame the inhibition caused by 5-azacytidine. L-Methionine on the other hand did not influence the L-tyrosine induction. Hydrocortisone brought about a tenfold rise of the activity of the TAT (Table 2). 5-Azacytidine caused decreased which could be completely overcome by L-methionine. TAT can be induced by tryptophan, too (Kriiger and G&z, 1980; Kriiger et al., 1968). There was only a slight inhibition of the tryptophan induction of TAT by 5-azacytidine (Table 3). This could be reversed by L-methionine. We have analyzed the time course of the azacytidine effect as well. From Table 4 it can be seen that 8 and 12 hr after AZ administration a superinduction was observed. As in a previous study (Kriiger and Greuer, 1966), there was a marked potentiation of the steroid effect on the induction of TAT by L-tyrosine. This can also be seen in Table 5. 5-Azacytidine inhibited this effect. In the presence of L-methionine, the inhibition was almost non-existent. of 5-azacytidine Injuence adenoribosylation metabolism
(2)
on
the
NAD
From earlier results we supposed that the NAD adenoribosylation metabolism was involved in the process of TAT induction (Kriiger and G&z, 1978, 1980). Therefore we studied if the NAD synthesis and the activity of the ADPR synthetase were influenced by 5-azacytidine. In normal rats, there was an inhibition of the NAD synthesis caused by DL-tryptophan in the liver (Table 6). There was no effect on the NAD synthesis initiated by nicotinamide. In adrenalectomized rats, however, rhe
1387
H.
1388 Table
KRUGER et al.
I. Influence of S-azxytidine on the mduction of tvrosine aminotransferase caused by L-tyrosine in normal an&Is Treatment
pm01 p-hydroxyphenylpyruvic acid!mg protein/hr
n
L-tyrosine” (300 mg/kg)
5
3.38 i 0.93
L-tyrosine (300 mgjkg) 5-aracytldine (25 mg/kg)
4
I .08 f 0.27
L-tyrosme (300 q/kg) L-methionine (300 mg:kg)
4
3.09 * I .53
4
3.24 k 0.52
L-tyrosine (300 mg/kg) L-methmnine (300 mg/kg) 5-azacytidme (25 &kg) 5-azacytidine (25 mgikg)
4
0.85 ? 0.14
0.15MNaCl
4
0.35 * 0.03
(IOmljkg)
“Treatment over a period of 4 hr.
Table 2. Influence of 5.azacytidine on the induction of tyrosine aminotransferase caused bv hvdrocortisone in adrenalectomized animals pm01 p-hydroxyphenylpyruvic acid/mg protein/hr
Treatment
n
Hydrocortisone” (IO mg/kg)
4
3.93 f I .oo
Hydrocortisone (10 mg/kg) 5-azacytidine (25 mg/kg)
5
1.77 k 0.36
Hydrocortisone (IO mg/kg) 5-azacytidine (25 m/kg) L-methionine (300 mgikg)
5
3.80 + 1.02
5-azacytidine (25 mgjkg)
3
0.30 f 0.08
0. I5 M NaCl (10 ml/kg)
4
0.43 i 0.07
“Treatment over a period of 4 hr.
Table 3. Influence of 5-ardcytidine on the induction of tyrosine aminotransferase by tryptophan firno p-hydroxyphenylpyruwc acid/mg proteinjhr
n
Treatment oL-tryptophan” (300 mgjkg)
3
3.43 * 0. I9
tx-tryptophan (300 m&kg) 5-azacytidine (25 mgjkg)
4
2.57 * 0.88
oL-tryptophan (300 mgikg) 5.azacytidine (25 mg/kg) + L-methionine (300 mglkg)
4
3.49 * 0.99
0.15 M NaCl (I 0 ml:ke)
3
1.33 + 0.26
“Termination of the experiments after 4 hr
Table 4. Influence of 5.azacytidine on the induction of tyrosine aminotransferase 4hr
8 hr
l2hr
NaCl (0.15 M: IO ml/kg)
1.41” * 1.41 (4)
I .30 f 0.35 (5)
0.84 k 0.09 (4)
mtryptophan
2.75 i 2.00 (5)
7.07 * 0.34 (4)
1.95 * 0.55 (5)
1.55 i 0.51 (5)
3.31 f0.13 (5)
3.96 i I .48 (5)
Treatment
(500 mglkg)
5-azacytidine (25 mg/kg) wtryptophan (500 mg/kg) + S-azacytidine (25 mg/kg)
3.08f0.17 8.65 + 2.32 (5) (5) The substances were applied intraperitoneally at times zero. “pm01 p-hydroxyphenylpyruvic acid/mg protein/hr.
NAD content increased if tryptophan was applied together with Sazacytidine (Table 7). We have also tested the influence on the activity of ADPR transferase. From Table 8 it can be seen that azacytidine inhibited ADPR transferase to some extent.
5.98 f I .94 (6)
DISCUSSION
Although there are many mechanisms by which gene expression can be controlled, the role of gene methylation is receiving wide attention. In some cases a methylated gene is inactive, while hypomethylation
1389
Azacytidine on amino transferase and NAD metabolism Table 5. Influenceof 5-azacytidine
on the induction of tyrosine aminotran5fe~ds~ substances in adrenalectomized rats
by various Treatment ._~ 5-azacytidine (25 mg/kg) r-tyrosine
pmol p-hydroxyphenyipyruvic acid/q protein/hr
__-__n
(300 mgjkg)
4
0.37 ?r 0.01
5
0.46 ? 0.16
(2.5 mg/kg)
5
I .88i: 0.65
I.-tyrosine (300mg/kg) Hydrocortisone (2.5 mgjkg)
4
4.85 _+0.88
4
I .4s
4
3.66 i: 2.35
5
0.5oco.12
Hydrocorrisone
L-tyrosine (300mg/kg) Hydrocortisone (2.5 mgjkg) S-aracytidine (25 m&kg) L-tyrosine (300 mg/kg) Hydrocortisone (2.5 mg/kg) S-azacytidine (25 mgikg) t.-methionine (300 mg/kg) 0.15 M Nafl” “Treatment
(10 ml/kg)
i 0.3
I
over a period of 4 hr
Table 6. NAD synthesis
in difl’erent organs of normal Liver
-.O.lSMNaCI”
(IOmljkg)
O.lSMNdCl 5-azacytidine
(lOml/kg) (25 mg/kg)
rats under the influence of 5-azacytidine Brain
Spleen
Kidney
0.33 1 0.05 (8)
0.29 i 0.03 (7)
0.67 k 0.03 (7)
0.78~0.1s (4)
0.28 i_ 0.02 (S)
0.34 * 0.05 (5)
0.75 * 0.13 (S)
._____ 0.81” & 0.07 (6)
Nicotinamide
(500 mg/kg)
2.57 f 0.35 (4)
0.42 + 0.06 (4)
0.51 + 0.05 (4)
I .2s
Nicotinamide S-azacytidine
(SO0 mg’kg) (25 mg/kg)
2.53 f 0.30 (4)
0.29 + 0.09 (5)
0.47 i 0.06 (5)
1.41 -f-0.33 (S)
(300 mg/kg)
1.39*0.10 (4)
0.30 + 0.01 (4)
0.31 i 0.08 (4)
0.90 IO.08 (4)
oL-tryptophan (300 mg/kg) S-azacytidine (25 mg:kg)
1.00~0.17 (5)
0.30 * 0.07 (5)
0.26 f 0.03 (S)
0.76 -f 0.12 (5)
DL-tryptophan
~e~ination of the experiments ‘flrnol,ig wt NAD + NADH,.
Table
7. NAD
after 4 hr.
synthesis
in different organs of adrenalectomized influence of S-atacytidine Liver
Spleen
rats under
the
Kidney
Nicotinamide
(SO0 mgikg)
2.51” IO.64 (3)
0.37 * 0.04 (4)
1.13io.11 (4)
rx-tryptophan
(300 n&kg)
o.E%*o.13 (3)
0.23 + 0.03 (4)
0.59 + 0.0s (4)
O.R6i: 0.20 (4)
0.32 IO.06 (4)
0.72 + 0.06 (4)
S-azacytidine
+ 0.05 (3)
(25 m&g)
5-azacytidine Nicotinamide
(25 mg/kg) (SO0 mg/kg)
2.43 + 0.16 (4)
0.42 i_ 0.05 (4)
I .44 +
S-azacytidine oL-tryptophan
(25 mgikg) (300 mg/kg)
l.l6&0.09 (4)
0.33 * o.oi (4)
0.87 & 0.05 (4)
0.15 M N&l*
(~Omi~kg)
0.7 I * 0.09 101
0.25 z 0.04 10%
0.64 jlo.19 (91
“pmol/g wt NAD + NADH,. hTermination of the experiments
0.25
(4)
after 4 hr.
is necessary for gene expression (Felsenfeld and McGhee, 1982). According to this hypothesis, the presence of AZ in the DNA would block the methylation and concomitant inactivation of the regulatory in the continued inhibition of genes, resulting mammalian differentiation (Bolander, 1983). Recent reports suggest that it may not be suf%cient (Wilk et at., 1982; Ott et ai., 1982). In this paper, it is demonstrated that the inhibition of the enzyme induction of tyrosine aminotransferase by 5-azacytidine does not take place in the presence
of methionine. Whether this is due to interference with the DNA methylation has to be further elucidated. On the other hand, an influence of AZ on the NAD synthesis provoked by the injection of tryptophan was seen. Interestingly by that the NAD content increased in the liver of adrenalectomized animals if both tryptophan and AZ were present. This could have been due to an interference with ADPR transferase because the same phenomenon was found with inhibitors of this enzyme (Kriiger and in preparation) and with G&z, manuscript
H. KRUGER et al.
1390 Table 8. Influence of S-azacytidine, m-tryptophan and nicotinamide on the activity of the ADPR transferase in liver nuclei from normal rats Treatme&
cpm/mg DNAh
oL-tryptophan (500 mg/kg) 5.azacytidine (25 mgjkg)
4117
oL-tryptophan
4161
(500 mg/kg)
JonesP.A. and Taylor S. M. (1980) Cellular differentiation,
Nicotinamide (500 mg:kg) 5-azacytidine (25 mg/kg)
4203
Nicotinamide (500 mg/kg)
4.455
5-azacytidine (25 mg/kg)
4128
0.15 M NaCl (lOml/kg)
4650
“Termination 2 hr after application. “Incubation with labelled NAD for 10 min.
D-galactosamine (Kriiger et al., 1983). Further are required to elucidate this point.
studies
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