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Decrease in the helix-coil transition temperature of calf-thymus DNA in the presence of ethylcarbamate
SHORT COMMUNICATIONS
615
with DNAase I (EC 3.1.4.5, pancreatic DNAase, Worthington). Details will be reported in a separate communication. This work was supported in part by grant C-3475 (C4) from the National Cancer Institute, U. S. Public Health Service.
Oncology-Hematology Section o/ the Department o/ Medicine, and the Department o/ Biochemistry, Indiana University Medical Center, Indianapolis, Ind. (U.S.A.)
M.
E.
HODES*
MARY K . SWENSON
1 M. LASKOWSKI, SR., in P. D. BOYER, ]7~. LARDY AND K. ~V[YRBACK,The Enzymes, Vol. 5, Acad e m i c Press, Inc., N e w York, 1961, p. 139. 2 ~][. SHIMOMURA AND ]V[. LASKOWSKI, SR., Biochim. Biophys. Acta, 26 (1957) 198. a j . F. KOERNER AND R. L. SINSHEIMER, J. Biol. Chem., 228 (1957) lO39. * E. R. M. KAY, N. I. SIMMONS AND A. L. DOUNCE, Federation Proc., IO (1951) 177.
Received June I5th, 1962 * L e u k e m i a Society Scholar.
Biochim. Biophys. Acta, 61 (1962) 6 1 2 - 6 1 5
SC 7047
Decrease in the helix-coil transition temperature of coN-thymus D N A in the presence of ethylcarbamate It was recently reported by B E L M A N , LEVINE AND TROLL1 that 2-amino-I-naphthol, the carcinogenic metabolite of 2-naphthylamine, as well as I-amino-2-naphthol (which is also carcinogenic) cause a "significant lowering" of the melting temperature (Tin) of salmon-sperm DNA and a change of the melting profile. Related noncarcinogenic compounds and 2-naphthylamine itself do not show this effect. The authors concluded that these observations supported "the notion that carcinogens, like mutagens, have DNA as a primary target". Therefore, it was of interest to test the effect of the carcinogen ethylcarbamate (urethan) on the Tm of DNA, to see whether this compound, structurally so different from the cyclic carcinogens and mutagens, would also lower the helix-coil transition temperature of DlqA. The possible relevance to carcinogenesis of such an effect could be tested by comparing the effect of ethylcarbamate with that of its non-carcinogenic homolog methylcarbamate~,3. Highly polymerized calf-thymus DNA obtained from the Worthington Biochemical Corp. was used for these studies. Ethylcarbamate and methylcarbamate (both Laboratory Reagent grade) were obtained from British Drug Houses Ltd. These carbamate preparations showed significant absorbancy at 260 m}~, the approximate molar absorbancy coefficients being o.i and 0.3 for ethyl- and methylcarbamates respectively. Experimental and control DNA solutions were run simultaneously against a blank containing the carbamate but without DNA. The increase in absorbancy at Biochim, Biophys. Acta, 61 (1962) 6 1 5 - 6 1 7
616
SHORT COMMUNICATIONS
26o m/z, due to the helix-coil transition of DNM, was followed using a Beckman DU spectrophotometer equipped with heating coils surrounding the cell compartment. The melting temperature (Tm -~ the temperature at the midpoint of the transition) 5 was calculated from the curves obtained. Ethylcarbamate caused a lowering of the Tm of DNA (&Tin) of 7-8°/mole (Fig. I) in the concentration range 0.2-2.0 M. At a concentration of 0.02 M, no effect of ethylcarbamate could be detected (Fig. 2). Methylcarbamate showed the same degree of lowering of the Tm as ethylcarbamate when both were tested at 0.2 M concentration (Fig. 3). The decrease in melting temperature of 7-8°/mole ethylcarbamate (Fig. I), while more than double that found to be caused by urea (3 °) by Ts'o et al.% is much less than the values of 17 ° and higher which they found for various purine and pyrimidine derivatives, the most potent of which, caffeine, causes a decrease in 15
10
E
/° /
0'.5 110 115 Final concentration of urethan (M)
2'.0
Fig. I. Lowering of the melting temperature of DNA by ethylcarbamate. The medium contained 25 #g/ml DNA, o.15 M NaC1, O.Ol5 M sodium citrate and 0.02 M phosphate buffer at pH 7.4-
0.12
P
O.Og o
0.06
<
0.03 8O
85
gO
D5
Temperature (°C)
Fig. 2. Melting profile of DNP, in presence of 0.02 M ethylcarbamate. G}, coincident control and experimental points; O, ethylcarbamate system; O, control system. Systems as described in Fig.i.
Biochim. Biophys. Acta, 61 (1962) 615-617
SHORT COMMUNICATIONS
617
0.12
S
O.Og 0.0~ <
0.0~ 0
80 Temperature (°C)
F i g . 3. Melting profiles o f D N A in t h e presence of 0 . 2 M c a r b a m a t e . - , controls; . .., ethylc a r b a m a t e ; - - - , m e t h y l c a r b a m a t e . S y s t e m s as described in F i g . I.
Tm of 60 °. (These authors also confirmed by conductance measurements that the addition of non-ionic substances did not change the ionic concentration of the test system enough to affect the Tm of DNA.) The magnitude of urethan's effect is seen to be small compared to several agents which have not been shown to be carcinogenic e.g. caffeine which proved non-carcinogenic in a small scale test 7. At a concentration of 0.02 M ethylcarbamate, which can be obtained in a mouse following a carcinogenic (I rag/g) dose of urethanS,% there is no detectable effect of ethylcarbamate on the Tm of DNA. This fact, combined with the observation that the non-carcinogenic methylcarbamate 2,3 has tile same activity as ethylcarbamate (when tested at 0.2 N where an ethylcarbamate effect can be detected), does not support the idea that urethan carcinogenesis is due to a direct interaction with D N A resulting in a lowering of its melting temperature nor the generalization that all carcinogens "have D N A as a primary target". This research was supported in part by a U. S. Public Health Service research grant C-5263. The author is the Herbert Sidebothaln Research Associate of the Weizmann Institute of Science. The experiments were performed with the skilled technical assistance of A. L. SUSSWEIN.
Department o~ Experimental Biology, The Weizmann Inst#ute o/ Science, Rehovoth (Israel)
A. M. KAYE
1 S. BELMAN, E . LEVINE AND W . TROLL, Federation Proc., 21 (1962) 3 7 4 2 C. D . LARSEN, J. Natl. Cancer Inst., 8 (1947) 99. 3 I. ]~ERENBLUM, D . BEN-ISHAI, IX[. I~ARAN-GHERA, i . LAPIDOT, E . SIMON AND •. TRAININ, Biochem. Pharmacol., 2 (1959) 168. P. DOTY, Proc. Natl. Acad. Sci. U.S., 42 (1956) 791. 5 j. MARMUR AND P . DOTY, Nature, 183 (1959) 1 4 2 7 . 6 p . O . P . T s ' o , G . I~. HELMKAMP AND C. SANDER, Proc. Natl..4cad. Sei. U.S., 48 (1962) 6 8 6 . 7 L. L . BOUGHTON AND O . O . STOLAND, J. Am. Pharm. Ass., 32 (1943) 187. s E . BOYLAND AND E . RHODEN, Biochem. J., 4 4 (1949) 5 2 8 . 9 A . M. •AYE, Cancer Research, 2 0 (196o) 237.
Received July 23rd, 1962 Biochim. Biophys. Acta, 61 (1962) 6 1 5 - 6 1 7
615
with DNAase I (EC 3.1.4.5, pancreatic DNAase, Worthington). Details will be reported in a separate communication. This work was supported in part by grant C-3475 (C4) from the National Cancer Institute, U. S. Public Health Service.
Oncology-Hematology Section o/ the Department o/ Medicine, and the Department o/ Biochemistry, Indiana University Medical Center, Indianapolis, Ind. (U.S.A.)
M.
E.
HODES*
MARY K . SWENSON
1 M. LASKOWSKI, SR., in P. D. BOYER, ]7~. LARDY AND K. ~V[YRBACK,The Enzymes, Vol. 5, Acad e m i c Press, Inc., N e w York, 1961, p. 139. 2 ~][. SHIMOMURA AND ]V[. LASKOWSKI, SR., Biochim. Biophys. Acta, 26 (1957) 198. a j . F. KOERNER AND R. L. SINSHEIMER, J. Biol. Chem., 228 (1957) lO39. * E. R. M. KAY, N. I. SIMMONS AND A. L. DOUNCE, Federation Proc., IO (1951) 177.
Received June I5th, 1962 * L e u k e m i a Society Scholar.
Biochim. Biophys. Acta, 61 (1962) 6 1 2 - 6 1 5
SC 7047
Decrease in the helix-coil transition temperature of coN-thymus D N A in the presence of ethylcarbamate It was recently reported by B E L M A N , LEVINE AND TROLL1 that 2-amino-I-naphthol, the carcinogenic metabolite of 2-naphthylamine, as well as I-amino-2-naphthol (which is also carcinogenic) cause a "significant lowering" of the melting temperature (Tin) of salmon-sperm DNA and a change of the melting profile. Related noncarcinogenic compounds and 2-naphthylamine itself do not show this effect. The authors concluded that these observations supported "the notion that carcinogens, like mutagens, have DNA as a primary target". Therefore, it was of interest to test the effect of the carcinogen ethylcarbamate (urethan) on the Tm of DNA, to see whether this compound, structurally so different from the cyclic carcinogens and mutagens, would also lower the helix-coil transition temperature of DlqA. The possible relevance to carcinogenesis of such an effect could be tested by comparing the effect of ethylcarbamate with that of its non-carcinogenic homolog methylcarbamate~,3. Highly polymerized calf-thymus DNA obtained from the Worthington Biochemical Corp. was used for these studies. Ethylcarbamate and methylcarbamate (both Laboratory Reagent grade) were obtained from British Drug Houses Ltd. These carbamate preparations showed significant absorbancy at 260 m}~, the approximate molar absorbancy coefficients being o.i and 0.3 for ethyl- and methylcarbamates respectively. Experimental and control DNA solutions were run simultaneously against a blank containing the carbamate but without DNA. The increase in absorbancy at Biochim, Biophys. Acta, 61 (1962) 6 1 5 - 6 1 7
616
SHORT COMMUNICATIONS
26o m/z, due to the helix-coil transition of DNM, was followed using a Beckman DU spectrophotometer equipped with heating coils surrounding the cell compartment. The melting temperature (Tm -~ the temperature at the midpoint of the transition) 5 was calculated from the curves obtained. Ethylcarbamate caused a lowering of the Tm of DNA (&Tin) of 7-8°/mole (Fig. I) in the concentration range 0.2-2.0 M. At a concentration of 0.02 M, no effect of ethylcarbamate could be detected (Fig. 2). Methylcarbamate showed the same degree of lowering of the Tm as ethylcarbamate when both were tested at 0.2 M concentration (Fig. 3). The decrease in melting temperature of 7-8°/mole ethylcarbamate (Fig. I), while more than double that found to be caused by urea (3 °) by Ts'o et al.% is much less than the values of 17 ° and higher which they found for various purine and pyrimidine derivatives, the most potent of which, caffeine, causes a decrease in 15
10
E
/° /
0'.5 110 115 Final concentration of urethan (M)
2'.0
Fig. I. Lowering of the melting temperature of DNA by ethylcarbamate. The medium contained 25 #g/ml DNA, o.15 M NaC1, O.Ol5 M sodium citrate and 0.02 M phosphate buffer at pH 7.4-
0.12
P
O.Og o
0.06
<
0.03 8O
85
gO
D5
Temperature (°C)
Fig. 2. Melting profile of DNP, in presence of 0.02 M ethylcarbamate. G}, coincident control and experimental points; O, ethylcarbamate system; O, control system. Systems as described in Fig.i.
Biochim. Biophys. Acta, 61 (1962) 615-617
SHORT COMMUNICATIONS
617
0.12
S
O.Og 0.0~ <
0.0~ 0
80 Temperature (°C)
F i g . 3. Melting profiles o f D N A in t h e presence of 0 . 2 M c a r b a m a t e . - , controls; . .., ethylc a r b a m a t e ; - - - , m e t h y l c a r b a m a t e . S y s t e m s as described in F i g . I.
Tm of 60 °. (These authors also confirmed by conductance measurements that the addition of non-ionic substances did not change the ionic concentration of the test system enough to affect the Tm of DNA.) The magnitude of urethan's effect is seen to be small compared to several agents which have not been shown to be carcinogenic e.g. caffeine which proved non-carcinogenic in a small scale test 7. At a concentration of 0.02 M ethylcarbamate, which can be obtained in a mouse following a carcinogenic (I rag/g) dose of urethanS,% there is no detectable effect of ethylcarbamate on the Tm of DNA. This fact, combined with the observation that the non-carcinogenic methylcarbamate 2,3 has tile same activity as ethylcarbamate (when tested at 0.2 N where an ethylcarbamate effect can be detected), does not support the idea that urethan carcinogenesis is due to a direct interaction with D N A resulting in a lowering of its melting temperature nor the generalization that all carcinogens "have D N A as a primary target". This research was supported in part by a U. S. Public Health Service research grant C-5263. The author is the Herbert Sidebothaln Research Associate of the Weizmann Institute of Science. The experiments were performed with the skilled technical assistance of A. L. SUSSWEIN.
Department o~ Experimental Biology, The Weizmann Inst#ute o/ Science, Rehovoth (Israel)
A. M. KAYE
1 S. BELMAN, E . LEVINE AND W . TROLL, Federation Proc., 21 (1962) 3 7 4 2 C. D . LARSEN, J. Natl. Cancer Inst., 8 (1947) 99. 3 I. ]~ERENBLUM, D . BEN-ISHAI, IX[. I~ARAN-GHERA, i . LAPIDOT, E . SIMON AND •. TRAININ, Biochem. Pharmacol., 2 (1959) 168. P. DOTY, Proc. Natl. Acad. Sci. U.S., 42 (1956) 791. 5 j. MARMUR AND P . DOTY, Nature, 183 (1959) 1 4 2 7 . 6 p . O . P . T s ' o , G . I~. HELMKAMP AND C. SANDER, Proc. Natl..4cad. Sei. U.S., 48 (1962) 6 8 6 . 7 L. L . BOUGHTON AND O . O . STOLAND, J. Am. Pharm. Ass., 32 (1943) 187. s E . BOYLAND AND E . RHODEN, Biochem. J., 4 4 (1949) 5 2 8 . 9 A . M. •AYE, Cancer Research, 2 0 (196o) 237.
Received July 23rd, 1962 Biochim. Biophys. Acta, 61 (1962) 6 1 5 - 6 1 7