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Differential inactivation of transforming DNA in vitro and in vivo by 4-hydroxyaminoquinoline 1-oxide
Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
193
D I F F E R E N T I A L I N A C T I V A T I O N OF T R A N S F O R M I N G DNA I N AND I N V I V O BY 4 - H Y D R O X Y A M I N O Q U I N O L I N E I - O X I D E
VITRO
Y U T A K A I S H I [ AND SOHE1 KONDO Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 (Japan) (Received May 25th, 1971)
SUMMARY
An Hcr strain of Bacillus subtilis showed about Io-fold higher sensitivity to killing by 4-hydroxyamincquinoline 1-oxide (4HAQO) than the parental Hcr+ strain, and the same differential sensitivity was observed between the same pair of strains after UV irradiation or 4-nitroquinoline 1-oxide (4NQO) treatment. DNA extracted from H c r - cells treated with 4HAQO showed higher transforming activity when assayed on Hcr~ cells than on H c r - cells in parallel to the case of DNA extracted from 4NQO-treated cells. However, DNA treated in vitro with 4HAQO in buffer solution showed the same transforming activity on both Hcr+ and H c r - cells. These results indicate that 4HAQO damage induced in DNA in vivo but not in vitro is repairable by the excision-repair mechanism.
INTRODUCTION
4-Nitroquinoline 1-oxide (4NQO), a potent, water-soluble carcinogen 15, is one of the most widely used chemical carcinogens 3. It is also mutagenic in phage in vivo 7, in bacteria 17 and in yeast 12 and is capable of inducing prophage in Escherichia coli 2. This chemical is not reactive in vitro6,19,~5; it is reduced in vivo1% or in vitro by extracts from various organs of rat2L to 4-hydroxyaminoquinoline 1-oxide (4HAQO), 4aminoquinoline 1-oxide (4AQO) and 4-aminoquinoline (4AQ). Similar to 4NQO, 4HAQO is carcinogenic, lethal, nmtagenic and capable of inducing prophage, whereas 4AQO and 4AQ show none of these effects1,2,17, 22. Furthermore, 4HAQO inactivates transforming DNA and bacteriophage genomes in vitro6,~% and induces single-strand seissions in DNA in vitro 2'~, whereas 4NQO does not inactivate transfroming DNA and induces no strand breaks in vitro1% 25. These findings seem to support the currently popular model that 4HAQO is an active intermediate in 4NQO carcinogenesis 2,8,19,24. However, there are two findings which are not readily compatible with this model. Firstly, 4NQO-induced damage in E. coli is not pyrimidine dimersl°, ~1 but is Abbreviations: 4AQ, 4-aminoquinoline; 4AQO, 4-aminoquinoline 1-oxide; 4HAQO, 4-hydroxyaminoquinoline 1-oxide; 4NQO, 4-nitroquinoline 1-oxide. Mutation Res., 13 (1971) 193-198
194
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ISHI1, S. KONI)()
reparable by the excision-repair nlechanism a as efficiently as are pyrimidine directs:', '-'~ induced b y ultraviolet (254 nm) radiation. This conclusion is based on the findings" " (a) that an Hcr (defective in host cell reactivation ability due to Exc character l excision-repair deficient]) strain of E. coli is about 30 times more sensitive than its parental Hcr -+ strain to 4NQO as well as to UV with respect to both killing and basechange m u t a t i o n and (b) t h a t the 4NQO-induced lethal or mutagenic effect is reduced for the Hcr + strain (but not for the Hcr strain) when 4NQO-treated cells are held in nutrient liquid medium before plating on acriflavine-containing agar, as is the case with a UV-induced lethal or mutagenic effect. Similar UV mimetic characteristics of 4NQO have been reported for killing in Bacillus subtilis 4 and Salmonella t v p h i m u r i u m (ref. 27). On the other hand, single-strand breaks in D N A (the kind of damage claimed to be induced by 4 H A Q O in vitro 25) are not reparable by the excision-repair mechanism according to the currently accepted repair model '~. Secondly, free phage P22 inactivated by 4 H A Q O in vilro shows the same survival when plated on cells of an Hcr strain of S. t),phimuriurn as t h a t on cells of its parental Hcr 4 strain, whereas UVirradiated P22 shows much lower survival on Hcr cells than on Hcr * cells'aL This paper reports t h a t 4 H A Q O damage induced in transforming D N A of B. subtilis in vivo is reparable but t h a t such damage induced in vitro is not. MATERIALS AND METHODS
Strains of B. subtilis Marburg 168 ( T r p ) and its derivative hcr- 9 (Trp-) were obtained from Dr. S. OKUBO. Strain hcr-9 is H c r - and UV s (sensitive to killing b y UV radiation) 18. Inactivation of colony4orming ability was measured as follows: Cells from exponentially growing cultures in Y P G medium (yeast extract, 0.5 g; polypeptone, 5 g; glucose, 5 g; MgSO4 ' 7 H~O, 0.2 g; KH2PO4, 8.48 g; K O H , 2.26 g; trisodium citrate, 0. 5 g; (NH4)2SO4, I g; made up to i 1) were washed once and resuspended in M / I 5 phosphate buffer (pH 6.8). Samples taken from the buffer suspension were irradiated b y UV from two I 5 - W low-pressure mercury germicidal lamps at a dose rate of 8 erg/mm2/sec. Or, t h e y were added with 4 H A Q O or 4NQO to various final concentrations, incubated for 4 ° rain at 37 ° with vigorous shaking and then diluted Ioo-fold or more with buffer to terminate the chemical treatment. After treatments, survivals were assayed b y plating on peptone agar (polypeptone, IO g; NaC1, 2.5 g; beef extract, 4 g; agar, 15 g; made up to i 1). Inactivation of transforming D N A in vitro b y 4HAQO was assayed as follows: D N A was extracted using phenol 2° at p H 9 from an exponentially growing culture of a prototrophic strain, B. subtilis Marburg SBI 9. Samples of the extracted D N A were dissolved in phosphate buffer (pH 6.8) at a concentration of 50 ttg/ml, 4 H A Q O added to a final concentration of 500 tiM and the mixture then incubated at 37 ° with vigorous shaking. At intervals, aliquots were withdrawn and diluted 50 times with buffer at ice temperature to terminate the chemical treatment. For assaying the transforming activity, o.I-ml samples were taken from each of these diluted aliquots and mixed with o.9-ml suspensions of cells (at ca. 3 " Io8 cells/ml) of strain 168 or hcr- 9 in a competent phase. Colonies grown from tryptophan-independent transformants on SPIZIZEN'S 23 minimal medium were scored as a measure of the residual activity of the transforming DNA. Details of these procedures have been described previously 2~. Mutation Res., 1.3 (1971) 193 198
4HAQO INACTIVATIONOF TRANSFORMINGDNA
195
Inactivation of transforming DNA in vivo by 4HAQO or 4NQO was assayed as follows: A prototrophic transformant of strain hcr-9 was grown to a logarithmic phase. Then, samples of the culture were treated with 4HAQO or 4NQO at various concentrations as described above for assaying inactivation of colony-forming ability. DNA was extracted from each of these treated samples. Transforming activity in the extracted DNA was assayed at a concentration of o.I #g/ml with strain 168 or hcr- 9 as described above. RESULTS AND DISCUSSION
As will be seen from Fig. I, the sensitivity difference between Hcr+ and Hcrstrains of B. subtilis for inactivation of colony-forming ability b y 4HAQO is identical to that by 4NQO and very similar to the difference in UV sensitivity. This agrees with previous reports concerning E. colP -n, B. subtilis 4 and S. typhimuriun, W. Fig. 2 shows that transforming activities of DNA treated with 4HAQO in vitro and assayed on Hcr+ cells were equal to those on H c r - cells. However, as shown in Fig. 3, DNA extracted from H c r - cells which had been inactivated b y 4HAQO or 4NQO showed higher transforming activity when assayed on Hcr + cells than on H c r cells. It should be noted that transforming DNA of B. subtilis irradiated with UV either in vitro or in vivo shows equally higher activity on Hcr+ cells than on H c r - cells (refs. z 3 and I4). UV DOSE (ERG/MMz) 400 800 1200
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Fig. I. Fractions of colony formers at various doses of UV, 4NQO and 4HA_QO in s t r a i n 168 (open symbol) and strain hcr- 9 (solid symbol). Cells were exposed to UV at r o o m t e m p e r a t u r e or to 4NQO or 4 H A Q O at various concentrations at 37 ° for 4 ° rain. © and O, UV; O and I , 4NQO; and • , 4HAQO. Fig. 2. Residual t r a n s f o r m i n g activity of D N A t r e a t e d w i t h 4 H A Q O in vitro. T r a n s f o r m i n g D N A was treated with 50o # M 4 H A Q O in buffer at 37 ° for various times. A, strain 168; • , strain hcr-9 Mutation Res., 13 (1971) 193-198
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Fig. 3. Residual t r a n s f o r m i n g activity of D N A treated with 4NQO or 4HAQO i~ vivo. Transforming D N A was extracted from cells of a p r o t o t r o p h i c t r a n s f o r m a n t of her-9 which had been pretreated with 4NQO or 4 H A Q O at various concentrations at 37 ° for 4 ° rain. T r a n s f o r m i n g activities were assayed on cells of strain 168 (open symbol) and strain her-9 (solid symbol). and I!, 4NQO; A and • , 4HAQO.
Fig. I clearly shows that the differential lethality in Hcr* and Her-- strains aft(r UV irradiation was quantitatively equal except for the shoulder part of the UV effect curve to that after treatment i~t vivo with either 4NQO or 4HAQO. This finding strongly suggests that the lethal damage induced by 4HAQO is very similar to 4NQO induced damage and that it is a kind of DNA damage excisable like pyrimidine dimers. This conclusion is further supported by the results shown in Fig. 3 that the reparable fraction of DNA damage induced in vivo by 4HAQO is the same as tile reparable fraction of 4NQO-induced DNA damage, if we measure the reparable fraction as the difference in transforming activity between DNA assayed on Hcr~ cells and that on Her cells. On the other hand, Fig. 2 shows that lesions on DNA induced by 4HAQO in vitro are not reparable, as previously reported by YAMAMOTOet al. 27 with phage P22. Obviously, the mode of action of 4HAQO in inducing damage to DNA in vitro and in vivo must be the same or different. In order to explain Figs. 2 and 3 by the "same action" model, we must assume that unexcisable lesions (or more specifically, strand breaks) are the major cause of inactivation of DNA i~z vitro but that those made in vivo are mostly eliminated or repaired by an unknown mechanism (which must be effective only for the iJ.~ vivo lesions), so that excisable lesions can play the major role in the inactivation of DNA in. vivo. If unexcisable lesions are not the major cause of inactivation in vitro, excisable lesions should be able to express their reparability in the fraction of DNA pieces possessing excisable lesions but lacking, by chance, unexcisable ones. But this was not the case (Fig. 2). Therefore, if the "same action" model holds true, 4HAQO must inactivate DNA at a higher rate in vitro than in vivo. As shown in Figs. 2 and 3, however, the opposite results were obtained experimentally : the 37% survival dose was "50o # M • 38 rain" (treatment at 500 p M for 38 rain) and "IOO ,uM • 4 ° rain" for 4HAQO in vitro and in vivo, respectively (compare Fig. 2 with Fig. 3). Therefore, we conclude that the mode of inactivation of DNA by 4HAQO in vitro is different from that in vivo. Mutation Res., 13 (1971) 193 19 ,~
4HAQO INACTIVATION OF TRANSFORMING DNA
19 7
One of the simplest "differential action" models is that the enhanced activity of 4HAQO in vivo is due to the action of an unknown enzyme(s). If this is the case, the assumed enzyme(s) is expected to convert either 4HAQO or4NQO (or after enzymatic conversion to 4HAQ0) to the same unknown active intermediate(s) capable of inducing excisable damage to DNA, because DNA damage induced in vivo by 4HAQO and 4NQO showed the same pattern of repair kinetics. This is a working hypothesis under study in our laboratory. ACKNOWLEDGMENTS
This work was supported by Grants-in-Aid from the Ministry of Education, Japan. We thank Dr. S. OKUBO for kindly supplying the strains, and Miss N. MURAOKA for her technical advice. REFERENCES I E.xDo, H., AND F. KvME, I n d u c t i o n of s a r c o m a in r a t s [by s u b c u t a n e o u s injection of 4-hyd r o x y a m i n o q u i n o l i n e i-oxide, Naturwissenschaften, 5 ° (1963) 525-526. 2 ENDO, H., M. ISHIZAWA AND T. KAMIYA, I n d u c t i o n of b a c t e r i o p h a g e f o r m a t i o n in lysogenic b a c t e r i a b y a p o t e n t carcinogen, 4-nitroquinoline i-oxide, a n d its derivatives, Nature, 198 (1963) I 9 5 - I 9 6 . 3 ENDO, H., T. ONO AND T. SUGIMURA (Eds.), Chemistry and Biological Actions of 4-Nitroquinoline z-Oxide, Springer, Berlin, 1971 . 4 12ELKNER, I. C., AND F. I~ADLIJBAR, Parallel b e t w e e n u l t r a v i o l e t light a n d 4-nitroquinoline 1-oxide s e n s i t i v i t y in Bacillus subtilis, J. Bacteriol., 96 (1968) I 4 4 8 - I 4 4 9 . 5 HOW'ARD-FLANDERS, P., D N A repair, Ann. Rev. Biochem., 37 (1968) 175-2oo. 6 ISHIZAWA, M., AND H. t~NDO, O n t h e m o d e of action of a p o t e n t carcinogen, 4 - h y d r o x y l a m i n o quinoline i-oxide, on b a c t e r i o p h a g e T4, Biochem. Pharmacol., 16 (I967) 637-646. 7 ISHIZAWA, M., AND H. ENDO, M u t a g e n i c effect of a carcinogen, 4-nitroquinoline 1-oxide, in b a c t e r i o p h a g e T4, Mutation Res., 9 (197 o) 134-1378 K&WAZOE, Y., M. TACHIBANA, t~. AOKI AND W. NAKAHARA, T h e s t r u c t u r e c a r c i n o g e n i c i t y r e l a t i o n s h i p a n l o n g d e r i v a t i v e s of 4-nitro a n d 4 - h y d r o x y l a m i n o q u i n o l i n e i-oxides, Biochem. Pharmacol., 16 (1967) 631-636. 9 KONDO, S., M u t a g e n i c i t y versus r a d i o s e n s i t i v i t y in Escherichia coli, Proc. 221h Intern. Congr. Genetics, Tokyo, 2 (1968) 126-127. IO I~ONDO, S., AND T. IgATO, P h o t o r e a c t i v a t i o n of m u t a t i o n a n d killing in Escherichia coli, Advan. Biol. 3Ied. Phys., 12 (1968) 283-298. 11 KoNI~O, S., H. ICHIKAWA, I~. IWO AND T. KATO, B a s e - c h a n g e m u t a g e n e s i s a n d p r o p h a g e induction in s t r a i n s of Escherichia coli w i t h different D N A repair capacities, Genetics, 66 (197o) 187217. 12 MIFUCHI, I., M. H o s o I , Y. YANAGIHARA AND M. NISHIDA, Studies on t h e t o x o h o r m o n e - l i k e s u b s t a n c e s in t h e respiration-deficient m u t a n t of Saccharomyces cerevisiae i n d u c e d b y 4-nitroquinoline 1-oxide, Gann, 54 (1963) 205 211. 13 MUNAKATA, N., R e p a i r of u l t r a v i o l e t - i n d u c e d d a m a g e in t r a n s f o r m i n g D N A of Bacillus subtills, Japan. J. Genetics, 45 (197 o) 1-9. 14 MUNAKATA, N., AND Y. IKEDA, I n a c t i v a t i o n of t r a n s f o r m i n g D N A b y u l t r a v i o l e t irradiation: a s t u d y with u l t r a v i o l e t - s e n s i t i v e m u t a n t s of Bacillus subtilis, Mutation Res., 7 (1969) 133-139. 15 •AKAIIARA, W., F. FUKUDA AND T. SUGIMURA, Carcinogenic a c t i o n of 4-nitroquinoline Noxide, Gann, 48 (1957) 129-137. 16 OKABAYASHI, T., AND A. YOSHIMOTO, R e d u c t i o n of 4-nitroquinoline 1-oxide b y microo r g a n i s m s , Chem. Pharm. Bull. (Tokyo), IO (1962) 1221-1226. 17 OKABAYASHI, T., ix,~. IDE, A. YOSHIMOTO AND M. OTSUBO, M u t a g e n i c a c t i v i t y of 4-nitroq u i n o l i n e 1-oxide a n d 4 - h y d r o x y a m i n o q u i n o l i n e 1-oxide on bacteria, Chem. Pharm. Bull. (Tokyo), 13 (i965) 61o-611. 18 OKUBO, S., AND \V. R. ROMIG, I m p a i r e d t r a n s f o r n l a b i l i t y of Bacillus subtilis m u t a n t sensitive to m i t o m y c i n C a n d u l t r a v i o l e t radiation, J. Mol. Biol., 15 (I966) 440-454 . 19 O>-o, T., T h e effects of carcinogens on t h e biological a c t i v i t y of nucleic acids, Protein, Nucleic Acid, Enzyme (Tokyo), 9 (1964) 1122-1128 (in J a p a n e s e ) .
Mutation Res., 13 (1971) 193-198
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20 SAITO, H., AND ik. ~'¥[IURA, P r e p a r a t i o n of t r a n s f o r m i n g d e o x y r i b o n u c l e i c acid by phenol t r e a t m e n t , Biochim. Biophys. Acta, 72 (1963) 619-629. 21 SETLOW, R. B., C y c l o b u t a n e - t y p e p y r i m i d i n e dimers in polynucleotides, Science, I53 (1900) 379-386. 22 SHIRASU, Y., AND A. OHTA, A p r e l i m i n a r y note on t h e c a r c i n o g e n i c i t y of 4 - h y d r o x y a m i n o quinoline i-oxide, Gann, 54 (1963) 221-223. 23 SPIZlZEN, J., T r a n s f o r m a t i o n of b i o c h e m i c a l l y deficient s t r a i n s of Bacillus subtilis by deoxyribonucleate, Proc. Natl. Acad. Sci. (U.S.), 44 (I958) I°72-IO78. 24 SUGIMURA, T., K. OKABE AND H. ENDO, T h e m e t a b o l i s m of 4-nitroquinoline I-oxide, a carcinogen, I. Conversion of 4-nitroquinoline I-oxide to 4 - a m i n o q u i n o l i n e 1-oxide b y r a t liver e n z y m e , Gann, 56 (1965) 489-5Ol. 25 SUGIMURA, T., H. OTAKE AND T. MATSUSHIMA, Single s t r a n d scissions of D N A c a u s e d b y a carcinogen, 4 - h y d r o x y l a m i n o q u i n o l i n e 1-oxide, Nature, 218 (I968) 392. 26 TANOOKA, H., AND Y. SAKAKIBARA, R a d i o r e s i s t a n t n a t u r e s of t h e t r a n s f o r m i n g a c t i v i t y of D N A in bacterial spores, Biochim. Biophys. Acla, I55 (1968) I3O-I42. 27 YAMAMOTO, N., S. FUKUDA AND H. TAKEBE, Effect of a p o t e n t carcinogen, 4-nitroquinoline 1-oxide, a n d its r e d u c e d form, 4 - h y d r o x y l a m i n o q u i n o l i n e 1-oxide, on bacterial a n d bacteriop h a g e g e n o m e s , Cancer Res., 3 ° (197 o) 2532-2537 .
Mutation Res., 13 (1971) 193 198
193
D I F F E R E N T I A L I N A C T I V A T I O N OF T R A N S F O R M I N G DNA I N AND I N V I V O BY 4 - H Y D R O X Y A M I N O Q U I N O L I N E I - O X I D E
VITRO
Y U T A K A I S H I [ AND SOHE1 KONDO Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 (Japan) (Received May 25th, 1971)
SUMMARY
An Hcr strain of Bacillus subtilis showed about Io-fold higher sensitivity to killing by 4-hydroxyamincquinoline 1-oxide (4HAQO) than the parental Hcr+ strain, and the same differential sensitivity was observed between the same pair of strains after UV irradiation or 4-nitroquinoline 1-oxide (4NQO) treatment. DNA extracted from H c r - cells treated with 4HAQO showed higher transforming activity when assayed on Hcr~ cells than on H c r - cells in parallel to the case of DNA extracted from 4NQO-treated cells. However, DNA treated in vitro with 4HAQO in buffer solution showed the same transforming activity on both Hcr+ and H c r - cells. These results indicate that 4HAQO damage induced in DNA in vivo but not in vitro is repairable by the excision-repair mechanism.
INTRODUCTION
4-Nitroquinoline 1-oxide (4NQO), a potent, water-soluble carcinogen 15, is one of the most widely used chemical carcinogens 3. It is also mutagenic in phage in vivo 7, in bacteria 17 and in yeast 12 and is capable of inducing prophage in Escherichia coli 2. This chemical is not reactive in vitro6,19,~5; it is reduced in vivo1% or in vitro by extracts from various organs of rat2L to 4-hydroxyaminoquinoline 1-oxide (4HAQO), 4aminoquinoline 1-oxide (4AQO) and 4-aminoquinoline (4AQ). Similar to 4NQO, 4HAQO is carcinogenic, lethal, nmtagenic and capable of inducing prophage, whereas 4AQO and 4AQ show none of these effects1,2,17, 22. Furthermore, 4HAQO inactivates transforming DNA and bacteriophage genomes in vitro6,~% and induces single-strand seissions in DNA in vitro 2'~, whereas 4NQO does not inactivate transfroming DNA and induces no strand breaks in vitro1% 25. These findings seem to support the currently popular model that 4HAQO is an active intermediate in 4NQO carcinogenesis 2,8,19,24. However, there are two findings which are not readily compatible with this model. Firstly, 4NQO-induced damage in E. coli is not pyrimidine dimersl°, ~1 but is Abbreviations: 4AQ, 4-aminoquinoline; 4AQO, 4-aminoquinoline 1-oxide; 4HAQO, 4-hydroxyaminoquinoline 1-oxide; 4NQO, 4-nitroquinoline 1-oxide. Mutation Res., 13 (1971) 193-198
194
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ISHI1, S. KONI)()
reparable by the excision-repair nlechanism a as efficiently as are pyrimidine directs:', '-'~ induced b y ultraviolet (254 nm) radiation. This conclusion is based on the findings" " (a) that an Hcr (defective in host cell reactivation ability due to Exc character l excision-repair deficient]) strain of E. coli is about 30 times more sensitive than its parental Hcr -+ strain to 4NQO as well as to UV with respect to both killing and basechange m u t a t i o n and (b) t h a t the 4NQO-induced lethal or mutagenic effect is reduced for the Hcr + strain (but not for the Hcr strain) when 4NQO-treated cells are held in nutrient liquid medium before plating on acriflavine-containing agar, as is the case with a UV-induced lethal or mutagenic effect. Similar UV mimetic characteristics of 4NQO have been reported for killing in Bacillus subtilis 4 and Salmonella t v p h i m u r i u m (ref. 27). On the other hand, single-strand breaks in D N A (the kind of damage claimed to be induced by 4 H A Q O in vitro 25) are not reparable by the excision-repair mechanism according to the currently accepted repair model '~. Secondly, free phage P22 inactivated by 4 H A Q O in vilro shows the same survival when plated on cells of an Hcr strain of S. t),phimuriurn as t h a t on cells of its parental Hcr 4 strain, whereas UVirradiated P22 shows much lower survival on Hcr cells than on Hcr * cells'aL This paper reports t h a t 4 H A Q O damage induced in transforming D N A of B. subtilis in vivo is reparable but t h a t such damage induced in vitro is not. MATERIALS AND METHODS
Strains of B. subtilis Marburg 168 ( T r p ) and its derivative hcr- 9 (Trp-) were obtained from Dr. S. OKUBO. Strain hcr-9 is H c r - and UV s (sensitive to killing b y UV radiation) 18. Inactivation of colony4orming ability was measured as follows: Cells from exponentially growing cultures in Y P G medium (yeast extract, 0.5 g; polypeptone, 5 g; glucose, 5 g; MgSO4 ' 7 H~O, 0.2 g; KH2PO4, 8.48 g; K O H , 2.26 g; trisodium citrate, 0. 5 g; (NH4)2SO4, I g; made up to i 1) were washed once and resuspended in M / I 5 phosphate buffer (pH 6.8). Samples taken from the buffer suspension were irradiated b y UV from two I 5 - W low-pressure mercury germicidal lamps at a dose rate of 8 erg/mm2/sec. Or, t h e y were added with 4 H A Q O or 4NQO to various final concentrations, incubated for 4 ° rain at 37 ° with vigorous shaking and then diluted Ioo-fold or more with buffer to terminate the chemical treatment. After treatments, survivals were assayed b y plating on peptone agar (polypeptone, IO g; NaC1, 2.5 g; beef extract, 4 g; agar, 15 g; made up to i 1). Inactivation of transforming D N A in vitro b y 4HAQO was assayed as follows: D N A was extracted using phenol 2° at p H 9 from an exponentially growing culture of a prototrophic strain, B. subtilis Marburg SBI 9. Samples of the extracted D N A were dissolved in phosphate buffer (pH 6.8) at a concentration of 50 ttg/ml, 4 H A Q O added to a final concentration of 500 tiM and the mixture then incubated at 37 ° with vigorous shaking. At intervals, aliquots were withdrawn and diluted 50 times with buffer at ice temperature to terminate the chemical treatment. For assaying the transforming activity, o.I-ml samples were taken from each of these diluted aliquots and mixed with o.9-ml suspensions of cells (at ca. 3 " Io8 cells/ml) of strain 168 or hcr- 9 in a competent phase. Colonies grown from tryptophan-independent transformants on SPIZIZEN'S 23 minimal medium were scored as a measure of the residual activity of the transforming DNA. Details of these procedures have been described previously 2~. Mutation Res., 1.3 (1971) 193 198
4HAQO INACTIVATIONOF TRANSFORMINGDNA
195
Inactivation of transforming DNA in vivo by 4HAQO or 4NQO was assayed as follows: A prototrophic transformant of strain hcr-9 was grown to a logarithmic phase. Then, samples of the culture were treated with 4HAQO or 4NQO at various concentrations as described above for assaying inactivation of colony-forming ability. DNA was extracted from each of these treated samples. Transforming activity in the extracted DNA was assayed at a concentration of o.I #g/ml with strain 168 or hcr- 9 as described above. RESULTS AND DISCUSSION
As will be seen from Fig. I, the sensitivity difference between Hcr+ and Hcrstrains of B. subtilis for inactivation of colony-forming ability b y 4HAQO is identical to that by 4NQO and very similar to the difference in UV sensitivity. This agrees with previous reports concerning E. colP -n, B. subtilis 4 and S. typhimuriun, W. Fig. 2 shows that transforming activities of DNA treated with 4HAQO in vitro and assayed on Hcr+ cells were equal to those on H c r - cells. However, as shown in Fig. 3, DNA extracted from H c r - cells which had been inactivated b y 4HAQO or 4NQO showed higher transforming activity when assayed on Hcr + cells than on H c r cells. It should be noted that transforming DNA of B. subtilis irradiated with UV either in vitro or in vivo shows equally higher activity on Hcr+ cells than on H c r - cells (refs. z 3 and I4). UV DOSE (ERG/MMz) 400 800 1200
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0 1 2 3 TIME TREATED WITH 4HAQO
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Fig. I. Fractions of colony formers at various doses of UV, 4NQO and 4HA_QO in s t r a i n 168 (open symbol) and strain hcr- 9 (solid symbol). Cells were exposed to UV at r o o m t e m p e r a t u r e or to 4NQO or 4 H A Q O at various concentrations at 37 ° for 4 ° rain. © and O, UV; O and I , 4NQO; and • , 4HAQO. Fig. 2. Residual t r a n s f o r m i n g activity of D N A t r e a t e d w i t h 4 H A Q O in vitro. T r a n s f o r m i n g D N A was treated with 50o # M 4 H A Q O in buffer at 37 ° for various times. A, strain 168; • , strain hcr-9 Mutation Res., 13 (1971) 193-198
190
Z
~. fstIll, s. bi()Nl~(I
0.6
o 0.4 Z
g
iI
Ol 0
I00
4NQO OR 4HAO0 DOSE
200 (IuM'4OMIN)
Fig. 3. Residual t r a n s f o r m i n g activity of D N A treated with 4NQO or 4HAQO i~ vivo. Transforming D N A was extracted from cells of a p r o t o t r o p h i c t r a n s f o r m a n t of her-9 which had been pretreated with 4NQO or 4 H A Q O at various concentrations at 37 ° for 4 ° rain. T r a n s f o r m i n g activities were assayed on cells of strain 168 (open symbol) and strain her-9 (solid symbol). and I!, 4NQO; A and • , 4HAQO.
Fig. I clearly shows that the differential lethality in Hcr* and Her-- strains aft(r UV irradiation was quantitatively equal except for the shoulder part of the UV effect curve to that after treatment i~t vivo with either 4NQO or 4HAQO. This finding strongly suggests that the lethal damage induced by 4HAQO is very similar to 4NQO induced damage and that it is a kind of DNA damage excisable like pyrimidine dimers. This conclusion is further supported by the results shown in Fig. 3 that the reparable fraction of DNA damage induced in vivo by 4HAQO is the same as tile reparable fraction of 4NQO-induced DNA damage, if we measure the reparable fraction as the difference in transforming activity between DNA assayed on Hcr~ cells and that on Her cells. On the other hand, Fig. 2 shows that lesions on DNA induced by 4HAQO in vitro are not reparable, as previously reported by YAMAMOTOet al. 27 with phage P22. Obviously, the mode of action of 4HAQO in inducing damage to DNA in vitro and in vivo must be the same or different. In order to explain Figs. 2 and 3 by the "same action" model, we must assume that unexcisable lesions (or more specifically, strand breaks) are the major cause of inactivation of DNA i~z vitro but that those made in vivo are mostly eliminated or repaired by an unknown mechanism (which must be effective only for the iJ.~ vivo lesions), so that excisable lesions can play the major role in the inactivation of DNA in. vivo. If unexcisable lesions are not the major cause of inactivation in vitro, excisable lesions should be able to express their reparability in the fraction of DNA pieces possessing excisable lesions but lacking, by chance, unexcisable ones. But this was not the case (Fig. 2). Therefore, if the "same action" model holds true, 4HAQO must inactivate DNA at a higher rate in vitro than in vivo. As shown in Figs. 2 and 3, however, the opposite results were obtained experimentally : the 37% survival dose was "50o # M • 38 rain" (treatment at 500 p M for 38 rain) and "IOO ,uM • 4 ° rain" for 4HAQO in vitro and in vivo, respectively (compare Fig. 2 with Fig. 3). Therefore, we conclude that the mode of inactivation of DNA by 4HAQO in vitro is different from that in vivo. Mutation Res., 13 (1971) 193 19 ,~
4HAQO INACTIVATION OF TRANSFORMING DNA
19 7
One of the simplest "differential action" models is that the enhanced activity of 4HAQO in vivo is due to the action of an unknown enzyme(s). If this is the case, the assumed enzyme(s) is expected to convert either 4HAQO or4NQO (or after enzymatic conversion to 4HAQ0) to the same unknown active intermediate(s) capable of inducing excisable damage to DNA, because DNA damage induced in vivo by 4HAQO and 4NQO showed the same pattern of repair kinetics. This is a working hypothesis under study in our laboratory. ACKNOWLEDGMENTS
This work was supported by Grants-in-Aid from the Ministry of Education, Japan. We thank Dr. S. OKUBO for kindly supplying the strains, and Miss N. MURAOKA for her technical advice. REFERENCES I E.xDo, H., AND F. KvME, I n d u c t i o n of s a r c o m a in r a t s [by s u b c u t a n e o u s injection of 4-hyd r o x y a m i n o q u i n o l i n e i-oxide, Naturwissenschaften, 5 ° (1963) 525-526. 2 ENDO, H., M. ISHIZAWA AND T. KAMIYA, I n d u c t i o n of b a c t e r i o p h a g e f o r m a t i o n in lysogenic b a c t e r i a b y a p o t e n t carcinogen, 4-nitroquinoline i-oxide, a n d its derivatives, Nature, 198 (1963) I 9 5 - I 9 6 . 3 ENDO, H., T. ONO AND T. SUGIMURA (Eds.), Chemistry and Biological Actions of 4-Nitroquinoline z-Oxide, Springer, Berlin, 1971 . 4 12ELKNER, I. C., AND F. I~ADLIJBAR, Parallel b e t w e e n u l t r a v i o l e t light a n d 4-nitroquinoline 1-oxide s e n s i t i v i t y in Bacillus subtilis, J. Bacteriol., 96 (1968) I 4 4 8 - I 4 4 9 . 5 HOW'ARD-FLANDERS, P., D N A repair, Ann. Rev. Biochem., 37 (1968) 175-2oo. 6 ISHIZAWA, M., AND H. t~NDO, O n t h e m o d e of action of a p o t e n t carcinogen, 4 - h y d r o x y l a m i n o quinoline i-oxide, on b a c t e r i o p h a g e T4, Biochem. Pharmacol., 16 (I967) 637-646. 7 ISHIZAWA, M., AND H. ENDO, M u t a g e n i c effect of a carcinogen, 4-nitroquinoline 1-oxide, in b a c t e r i o p h a g e T4, Mutation Res., 9 (197 o) 134-1378 K&WAZOE, Y., M. TACHIBANA, t~. AOKI AND W. NAKAHARA, T h e s t r u c t u r e c a r c i n o g e n i c i t y r e l a t i o n s h i p a n l o n g d e r i v a t i v e s of 4-nitro a n d 4 - h y d r o x y l a m i n o q u i n o l i n e i-oxides, Biochem. Pharmacol., 16 (1967) 631-636. 9 KONDO, S., M u t a g e n i c i t y versus r a d i o s e n s i t i v i t y in Escherichia coli, Proc. 221h Intern. Congr. Genetics, Tokyo, 2 (1968) 126-127. IO I~ONDO, S., AND T. IgATO, P h o t o r e a c t i v a t i o n of m u t a t i o n a n d killing in Escherichia coli, Advan. Biol. 3Ied. Phys., 12 (1968) 283-298. 11 KoNI~O, S., H. ICHIKAWA, I~. IWO AND T. KATO, B a s e - c h a n g e m u t a g e n e s i s a n d p r o p h a g e induction in s t r a i n s of Escherichia coli w i t h different D N A repair capacities, Genetics, 66 (197o) 187217. 12 MIFUCHI, I., M. H o s o I , Y. YANAGIHARA AND M. NISHIDA, Studies on t h e t o x o h o r m o n e - l i k e s u b s t a n c e s in t h e respiration-deficient m u t a n t of Saccharomyces cerevisiae i n d u c e d b y 4-nitroquinoline 1-oxide, Gann, 54 (1963) 205 211. 13 MUNAKATA, N., R e p a i r of u l t r a v i o l e t - i n d u c e d d a m a g e in t r a n s f o r m i n g D N A of Bacillus subtills, Japan. J. Genetics, 45 (197 o) 1-9. 14 MUNAKATA, N., AND Y. IKEDA, I n a c t i v a t i o n of t r a n s f o r m i n g D N A b y u l t r a v i o l e t irradiation: a s t u d y with u l t r a v i o l e t - s e n s i t i v e m u t a n t s of Bacillus subtilis, Mutation Res., 7 (1969) 133-139. 15 •AKAIIARA, W., F. FUKUDA AND T. SUGIMURA, Carcinogenic a c t i o n of 4-nitroquinoline Noxide, Gann, 48 (1957) 129-137. 16 OKABAYASHI, T., AND A. YOSHIMOTO, R e d u c t i o n of 4-nitroquinoline 1-oxide b y microo r g a n i s m s , Chem. Pharm. Bull. (Tokyo), IO (1962) 1221-1226. 17 OKABAYASHI, T., ix,~. IDE, A. YOSHIMOTO AND M. OTSUBO, M u t a g e n i c a c t i v i t y of 4-nitroq u i n o l i n e 1-oxide a n d 4 - h y d r o x y a m i n o q u i n o l i n e 1-oxide on bacteria, Chem. Pharm. Bull. (Tokyo), 13 (i965) 61o-611. 18 OKUBO, S., AND \V. R. ROMIG, I m p a i r e d t r a n s f o r n l a b i l i t y of Bacillus subtilis m u t a n t sensitive to m i t o m y c i n C a n d u l t r a v i o l e t radiation, J. Mol. Biol., 15 (I966) 440-454 . 19 O>-o, T., T h e effects of carcinogens on t h e biological a c t i v i t y of nucleic acids, Protein, Nucleic Acid, Enzyme (Tokyo), 9 (1964) 1122-1128 (in J a p a n e s e ) .
Mutation Res., 13 (1971) 193-198
198
v. ISHII, S. KONI)(~
20 SAITO, H., AND ik. ~'¥[IURA, P r e p a r a t i o n of t r a n s f o r m i n g d e o x y r i b o n u c l e i c acid by phenol t r e a t m e n t , Biochim. Biophys. Acta, 72 (1963) 619-629. 21 SETLOW, R. B., C y c l o b u t a n e - t y p e p y r i m i d i n e dimers in polynucleotides, Science, I53 (1900) 379-386. 22 SHIRASU, Y., AND A. OHTA, A p r e l i m i n a r y note on t h e c a r c i n o g e n i c i t y of 4 - h y d r o x y a m i n o quinoline i-oxide, Gann, 54 (1963) 221-223. 23 SPIZlZEN, J., T r a n s f o r m a t i o n of b i o c h e m i c a l l y deficient s t r a i n s of Bacillus subtilis by deoxyribonucleate, Proc. Natl. Acad. Sci. (U.S.), 44 (I958) I°72-IO78. 24 SUGIMURA, T., K. OKABE AND H. ENDO, T h e m e t a b o l i s m of 4-nitroquinoline I-oxide, a carcinogen, I. Conversion of 4-nitroquinoline I-oxide to 4 - a m i n o q u i n o l i n e 1-oxide b y r a t liver e n z y m e , Gann, 56 (1965) 489-5Ol. 25 SUGIMURA, T., H. OTAKE AND T. MATSUSHIMA, Single s t r a n d scissions of D N A c a u s e d b y a carcinogen, 4 - h y d r o x y l a m i n o q u i n o l i n e 1-oxide, Nature, 218 (I968) 392. 26 TANOOKA, H., AND Y. SAKAKIBARA, R a d i o r e s i s t a n t n a t u r e s of t h e t r a n s f o r m i n g a c t i v i t y of D N A in bacterial spores, Biochim. Biophys. Acla, I55 (1968) I3O-I42. 27 YAMAMOTO, N., S. FUKUDA AND H. TAKEBE, Effect of a p o t e n t carcinogen, 4-nitroquinoline 1-oxide, a n d its r e d u c e d form, 4 - h y d r o x y l a m i n o q u i n o l i n e 1-oxide, on bacterial a n d bacteriop h a g e g e n o m e s , Cancer Res., 3 ° (197 o) 2532-2537 .
Mutation Res., 13 (1971) 193 198