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Mutagenicity of 10-methylated acridines for bacteriophage T4
M u t a t i o n Research Elsevier Publishing Company, Printed in The Netherlands
227
Amsterdam
Mutagenicity of lO-methylated acridines for bacteriophage T4 LERMAN5 reported that methylation of the ring nitrogen of weakly mutagenic acridine and strongly mutagenic 9-aminoacridine abolished mutagenic activity. The methylated derivatives were found, however, to bind in vitro to DNA at least as effectively as the parent compounds 5,7. This lack of correspondence between mutagenic and intercalating activities led LERMAN to conclude, tentatively, that intercalation m a y be a necessary but insufficient condition for mutagenesis. We now report that 9-amino-zo-methylacridinium is mutagenic for bacteriophage T4 r I I mutants. 9-Amino-Io-methylacridinium iodide was prepared from 9-aminoacridine ~ and separated from the parent compound bv chromatography on a column of silica gel (lOO-2OO mesh, 4.5 cm diam. × 46 cm for I5o-mg sample) using butan-I-ol:5 N acetic acid 7 : 3 v/v3. The product was further purified by preparative thin-layer chromatography on silica gel using the same solvent, and gave a single spot RF -- 0.35. In this system 9-aminoacridinium has Re = 0.52. The purified product was eluted with water and shown to be 9-amino-Io-methylacridinium by comparison of the absorption spectrum in methanol and in methanol containing I N K O H with published data 1. TABLE
I
ACRIDINt~-INDUCF.D AND BRENNER 6
Phage
REVERSION
OF
T 4 rlI MUTANTS
A gent
ACI5
AND
Test conc. M. io ~
ACI5
--
Acridine orange 9-amino- I o-methylacridinium Proflavine 9-aminoacridine Acridine yellow
2.o iodide
iodide
Reversion f r e q u e n c y • 1o s 0. 7
5.4
1.7
I OOO
1,9 2,0 2.o
3 3°0 5000 14ooo
P43 9-an~ino- I o-methylacridinium
P 4 3 TESTED ACCORDING TO ()RGEL
o.I 7 o. 79 1 ..55
o.3 2. 3 z 7° 2 300
Bacteriophage T 4 mutants r l I ACI 5 and P43 and procedures used for testing reversion of these mutants were described by ORGEL AND BRENNER6. The results given in Table ! are essentially in agreement with published information except for 9-amino-Io-methylacridinium which increased the reversion frequency of both A C I 5 and P43. There was a steep dependence of reversion frequency on concentration of the acridine derivative for P43 - about Io3-fold for a Io-fold increase in concentration. LERMAN (loc. cit.) tested 9-amino-Io-methylacridinium at a m a x i m u m concentration about IOO × greater than the highest concentration used by us and found no mutagenic activity. However, he provided no data bearing on the identity of the substance he tested. The substance tested by us was shown to be free of 9-aminoacridine and to correspond in its absorption spectrum to published data for 9-aminoIo-methyl acridinium. The absorption spectrum in alkaline methanol provides particularly convincing evidence for the identity of tile tested material with 9-aminoIo-methylacridinium because this substance is converted to 9-amino-Io-methylacridan. 3 1 u t a t i o n R e s . , 15 (1972) 227 2 2 8
228
SHORT COMMUNICATIONS
In biological testing of ring-N-methylated acridines, account has to be taken of the fact that quaternary acridines are subject to nucleophilic attack at position 9. Hydroxyl ions or sulfydryl groups can react at that position to give derivatives of acridan. If position 9 is unsubstituted, further reaction may occur, especially if oxygen is present. Hydroxyl ions even at 2o ° and pH 7 react rapidly with I, 2 or 9 isomers of amino-Io-methylacridinium ion to give sparingly soluble carbinols but the reaction may be reversed with change in pH (reviewed by ALBEI~T~). The mutagenicity of 9-amino-Io-methylacridinium may be due to the substance itself, but it is not excluded that it is a derivative of the substance that is biologically active. The possible reactivity of quaternary acridines with biological constituents in vivo probably invalidates attempts to correlate their mutagenicity with their ability to intercalate in DNA in vitro. The demonstration that 9-amino-xo-methylacridinium is mutagenic, for whatever reason, may further invalidate even the tentative conclusion already drawn'. We thank Dr. S. BRENNER for a gift of the mutants and the National Cancer Institute of Canada and Medical Research Council of Canada for funds in support of this work.
Cancer Research Laboratory, University of Western Ontario, Londo:z 7 2 (Canada)
N . KADOHAMA
B. MUHLSTOCK J . A. MCCARTER
I ACIIESON, R. ?tL, M. L. BURSTALL, C. \¥. JEFFORI) AND I3. F. SAMSON, Some t a u t o m e r i c acridines, J. Chem. Soc., 0954) 3742-3746. 2 ALBERT, A., The ,4cridines, Arnold, L o n d o n , 211(1 ed., I960, pp. 332 343. 3 ALBERT, A., ibid., p. 15o. 4 ALBERT, A., ANt) B. RITCHIE, T h e n a t u r e of t h e a m i n o g r o u p in aminoacridincs, P a r t 11. Evidence from chemical reactions, J. Chem. Soc., (~943) 458-462. 5 LERMAN, L. S., Acridine m u t a g e n s a n d D N A s t r u c t u r e , ./r. Cell. Comp. PhysioL, 64, Suppl. t (I964) I - r 8 . 6 0 R G E L , A., AND S. HREXNER, M u t a g e n e s i s of b a c t e r i o p h a g e T 4 by acridines, ./. 3]oI. l~ird., 3 (I96i) 768. 7 IedVA, S. C., I n t e r a c t i o n s of m e t h y l a t e d acridines with DNA, Biochem. Biophys. lees. Commun., 23 (~966) 6 o 6 - 6 i r .
Received September 2Ist, I97I Revision received March 2Ist, I972 ..]lutation Res., 15 (T972) 227-228
227
Amsterdam
Mutagenicity of lO-methylated acridines for bacteriophage T4 LERMAN5 reported that methylation of the ring nitrogen of weakly mutagenic acridine and strongly mutagenic 9-aminoacridine abolished mutagenic activity. The methylated derivatives were found, however, to bind in vitro to DNA at least as effectively as the parent compounds 5,7. This lack of correspondence between mutagenic and intercalating activities led LERMAN to conclude, tentatively, that intercalation m a y be a necessary but insufficient condition for mutagenesis. We now report that 9-amino-zo-methylacridinium is mutagenic for bacteriophage T4 r I I mutants. 9-Amino-Io-methylacridinium iodide was prepared from 9-aminoacridine ~ and separated from the parent compound bv chromatography on a column of silica gel (lOO-2OO mesh, 4.5 cm diam. × 46 cm for I5o-mg sample) using butan-I-ol:5 N acetic acid 7 : 3 v/v3. The product was further purified by preparative thin-layer chromatography on silica gel using the same solvent, and gave a single spot RF -- 0.35. In this system 9-aminoacridinium has Re = 0.52. The purified product was eluted with water and shown to be 9-amino-Io-methylacridinium by comparison of the absorption spectrum in methanol and in methanol containing I N K O H with published data 1. TABLE
I
ACRIDINt~-INDUCF.D AND BRENNER 6
Phage
REVERSION
OF
T 4 rlI MUTANTS
A gent
ACI5
AND
Test conc. M. io ~
ACI5
--
Acridine orange 9-amino- I o-methylacridinium Proflavine 9-aminoacridine Acridine yellow
2.o iodide
iodide
Reversion f r e q u e n c y • 1o s 0. 7
5.4
1.7
I OOO
1,9 2,0 2.o
3 3°0 5000 14ooo
P43 9-an~ino- I o-methylacridinium
P 4 3 TESTED ACCORDING TO ()RGEL
o.I 7 o. 79 1 ..55
o.3 2. 3 z 7° 2 300
Bacteriophage T 4 mutants r l I ACI 5 and P43 and procedures used for testing reversion of these mutants were described by ORGEL AND BRENNER6. The results given in Table ! are essentially in agreement with published information except for 9-amino-Io-methylacridinium which increased the reversion frequency of both A C I 5 and P43. There was a steep dependence of reversion frequency on concentration of the acridine derivative for P43 - about Io3-fold for a Io-fold increase in concentration. LERMAN (loc. cit.) tested 9-amino-Io-methylacridinium at a m a x i m u m concentration about IOO × greater than the highest concentration used by us and found no mutagenic activity. However, he provided no data bearing on the identity of the substance he tested. The substance tested by us was shown to be free of 9-aminoacridine and to correspond in its absorption spectrum to published data for 9-aminoIo-methyl acridinium. The absorption spectrum in alkaline methanol provides particularly convincing evidence for the identity of tile tested material with 9-aminoIo-methylacridinium because this substance is converted to 9-amino-Io-methylacridan. 3 1 u t a t i o n R e s . , 15 (1972) 227 2 2 8
228
SHORT COMMUNICATIONS
In biological testing of ring-N-methylated acridines, account has to be taken of the fact that quaternary acridines are subject to nucleophilic attack at position 9. Hydroxyl ions or sulfydryl groups can react at that position to give derivatives of acridan. If position 9 is unsubstituted, further reaction may occur, especially if oxygen is present. Hydroxyl ions even at 2o ° and pH 7 react rapidly with I, 2 or 9 isomers of amino-Io-methylacridinium ion to give sparingly soluble carbinols but the reaction may be reversed with change in pH (reviewed by ALBEI~T~). The mutagenicity of 9-amino-Io-methylacridinium may be due to the substance itself, but it is not excluded that it is a derivative of the substance that is biologically active. The possible reactivity of quaternary acridines with biological constituents in vivo probably invalidates attempts to correlate their mutagenicity with their ability to intercalate in DNA in vitro. The demonstration that 9-amino-xo-methylacridinium is mutagenic, for whatever reason, may further invalidate even the tentative conclusion already drawn'. We thank Dr. S. BRENNER for a gift of the mutants and the National Cancer Institute of Canada and Medical Research Council of Canada for funds in support of this work.
Cancer Research Laboratory, University of Western Ontario, Londo:z 7 2 (Canada)
N . KADOHAMA
B. MUHLSTOCK J . A. MCCARTER
I ACIIESON, R. ?tL, M. L. BURSTALL, C. \¥. JEFFORI) AND I3. F. SAMSON, Some t a u t o m e r i c acridines, J. Chem. Soc., 0954) 3742-3746. 2 ALBERT, A., The ,4cridines, Arnold, L o n d o n , 211(1 ed., I960, pp. 332 343. 3 ALBERT, A., ibid., p. 15o. 4 ALBERT, A., ANt) B. RITCHIE, T h e n a t u r e of t h e a m i n o g r o u p in aminoacridincs, P a r t 11. Evidence from chemical reactions, J. Chem. Soc., (~943) 458-462. 5 LERMAN, L. S., Acridine m u t a g e n s a n d D N A s t r u c t u r e , ./r. Cell. Comp. PhysioL, 64, Suppl. t (I964) I - r 8 . 6 0 R G E L , A., AND S. HREXNER, M u t a g e n e s i s of b a c t e r i o p h a g e T 4 by acridines, ./. 3]oI. l~ird., 3 (I96i) 768. 7 IedVA, S. C., I n t e r a c t i o n s of m e t h y l a t e d acridines with DNA, Biochem. Biophys. lees. Commun., 23 (~966) 6 o 6 - 6 i r .
Received September 2Ist, I97I Revision received March 2Ist, I972 ..]lutation Res., 15 (T972) 227-228