BIOCHIMICA ET BIOPHYSICA ACTA
POLYCYCLIC AROMATIC H Y D R O C A R B O N S COVALENT B I N D I N G TO DNA AND EFFECTS ON T E M P L A T E FUNCTION E. W . C H A N AND J. K. B A L L
Cancer Research Laboratory, University o] Western Ontario, London, Ontario (Canada)
Exposure of solutions of DNA containing intercalated molecules of benz(a)pyrene or pyrene to y-irradiation resulted in very extensive covalent binding of these hydrocarbons to DNA. The extent of binding for benz(a) pyrene was I molecule for every IOO DNA nucleotides and for pyrene was I : 270 DNA nucleotides. It was also found that in vitro transcription was inhibited when DNA containing covalently bound benz(a)pyrene was used as template for Escherichia coli DNA dependent RNA polymerase.
One prevailing theory regarding the mechanism of chemical carcinogenesis proposes that the chemical carcinogens interact with the DNA of the affected cells, interfering with gene function thus initiating the cancer process 1. Such interactions of a covalent nature have been observed in vivo following administration of radioactively labeled polycyclic aromatic hydrocarbons to mice 2-5. Covalent binding of these carcinogens to DNA has also been demonstrated in vitro either by means of a liver microsomal enzyme system 6,7, or by induction with ionizing agents such as hydrogen peroxide s' 9, photo_10 and X-irradiation 11. The effects, if any, of these bound carcinogens on transcription of the DNA have not been demonstrated in a purified in vitro system. The major difficulty encountered in such an attempt is the very low level of binding obtained (I hydrocarbon: 5 ° 000-500 ooo DNA-P). We have been able to induce covalent binding of pyrene and benz(a)pyrene to DNA by means of v-irradiation to a much higher extent than has been reported (I : IOO DNA nucleotides for benz(a)pyrene and I : 270 DNA nucleotides for pyrene). Our preliminary results of RNA polymerase assays indicated that covalently bound benz (a)pyrene inhibited the transcription of the DNA template, Physical complexes between calf thymus DNA and pyrene or benz(a)pyrene were prepared in 2 • io-4 M cacodylate (pH 5.2) at a concentration of DNA of I rag/ ml (ref. 12). These complexes were exposed to 6°Co v-radiation in unsealed glass tubes in a v-cell (Atomic Energy of Canada Ltd., Ottawa, Canada). Lead shields were used to reduce the dose rate to about 29oorads/min. Following v-irradiation, the DNA: hydrocarbon complexes were precipitated b y the addition of 0. 5 M ammonium acetate and 3 vol. of cold IOO % ethanol. The precipitates were washed 3 times with cold ethanol and two times with ether and then redissolved in one-third of the original volume of lO -2 M NaC1 in lO -2 M Tris-HC1 (pH 7.9). As a control, the ethanol precipitation step was shown to remove virtually all the hydrocarbon ( < 99.9 %) from Biochim. Biophys. Acta, 238 (1971) 4 6 - 5 9
POLYC YCLIC AROMATIC HYDROCARBONS AND DNA
physical complexes which had not been irradiated. The extensive covalent linkage between these hydrocarbons and DNA achieved under the conditions used, enabled us to record the actual absorption spectra of the hydrocarbons covalently bound to the DNA. Fig. I shows there are further bathochromic shifts of the absorption peaks in
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Fig. I. A b s o r p t i o n spectra of covalent DNA: h y d r o c a r b o n complexes. The s p e c t r a of benz(a)pyrene (a) a n d of pyrene (b) in covalent complex with D N A ( ) were c o m p a r e d at equal c o n c e n t r a t i o n s w i t h those in physical complex with D!~A ( - - - ) and those in m e t h a n o l ( - . - ) . Spectra recorded in a Cary 15 recording s p e c t r o p h o t o m e t e r .
addition to the shifts which took place when the hydrocarbons were physically complexed to DNA. Fig. 2 shows the dose dependence of v-radiation induced covalent binding of the 020
Fig. 2. Effects of y-irradiation on t h e covalent binding of h y d r o c a r b o n s to DNA. Physical complexes of D N A with b e n z ( a ) p y r e n e (BP) a n d pyrene (P) (initial molar ratios h y d r o c a r b o n s : D1WA-P were i : 48 and i : 27, respectively) were exposed to various doses of y-radiation.Covalent complexes were isolated and their absorption spectra determined. The peak absorptions of t h e complexes are p l o t t e d against radiation dose. The points on t h e figure represent duplicate or triplicate irradiation experiments.
Biochim. Biophys. Acta, 238 (1971) 46-49
E . W . CHAN, J. K. BALL
hydrocarbons to DNA. The levels of binding rose sharply at radiation doses above 8 krads. The binding ratios which were computed from the concentrations of the DNA-P and of the unextractable hydrocarbons (by ethanol or cyclohexane) were I : IOO DNA nucleotides for benz(a)pyrene and I : 270 DNA nucleotides for pyrene at a dose of 17 krads. This binding ratio of benz(a)pyrene was about Ioo-fold higher t h a n that reported by RAPAPORT et al. ]1. The template activities of such covalent benz(a)pyrene : DNA complexes and of control irradiated DNA were compared in an in vitro RNA synthesizing system. It is evident from the time courses of synthesis in Fig. 3, there were essentially no
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15 30 INCUBATION
Fig. 3. Time courses of R N A synthesis directed b y control irradiated D N A ( Q - O ) and irradiated covalent b e n z I a ) p y r e n e : D N A complex ( x - - - x ) (molar ratio = I b e n z ( a ) p y r e n e : 37 ° D N A P). B o t h control D N A a n d D N A : b e n z ( a ) p y r e n e complex were exposed to the same dose of 7radiation (io krads) and were assayed in duplicate for t e m p l a t e activity in t h e s t a n d a r d R N A synthesis assay of CHAMBERLIN AND BERG le, using 80/~g of D N A and 20/*g of F r a c t i o n I V R N A polymerase isolated from E. coli B. Specific activity of the enzyme was a b o u t 22oo u n i t s / m g protein per h.
differences in the template activities of the control DNA and of the complex during the early incubation times (assayed at IO and 15 min). However, at later incubation times, the template activity of the complex became significantly lower than that of the control DNA. After 30 min of incubation the activity of the complex was only 80 % of t h a t of the control DNA. A similar degree of inhibition was found in two similar experiments using different preparations of enzyme and irradiated benz(a)pyrene : DNA complexes. The exposure of control DNA to io krads reduced its template activity b y approx. 45 %. Examination of the absorption spectrum of the complex in the incubation mixture at the end of the enzyme reaction, and after subsequent deproteinization of the mixture, confirmed that the benz (a)pyrene remained bound to the DNA throughout RNA synthesis. Comparisons of the hyperchromicities, heat-denaturation temperatures (Tin) and sedimentation profiles in a sucrose density gradient of the complex and control DNA, indicated there was no preferential denaturation or degradaBiochim. Biophys. Acta, 238 (1971) 46-49
POLYCYCLIC AROMATIC HYDROCARBONS AND D N A
tion of the complex due to y-irradiation. As RNA synthesis was carried out under conditions of low salt (o.o5 M KC1), it was likely that each enzyme molecule made only one RNA chain 13. In view of these considerations, the absence of any differences in the template activities of the control DNA and of the complex during early times, suggests to us that the benz(a)pyrene molecules in the complex neither block the binding of the RNA polymerase to the DNA template nor interfere with the initiation of transcription. As transcription proceeds along the template, some of the growing RNA chains presumably encountered a benz(a)pyrene molecule, and it was at this point where transcription was retarded or terminated. The extensive binding of the hydrocarbons to DNA achieved in the piesent work makes it feasible to study the mode and nature of the binding. The inhibitio on transcription observed here is in keeping with the known inhibitory effects of carcinogenic polycyclic aromatic hydrocarbons on RNA synthesis in vivo 14,1s. The present purified in vitro system, with some modifications, would permit the elucidation of the mechanism of inhibition. However, the significance of the inhibitory effect on transcription in relation to the mechanism(s) of chemical carcinogenesis has yet to be established.
We wish to thank Professor J. A. McCarter for helpful suggestions and discussion. This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada from which one of us (E.W.C.) received a predoctoral fellowship.
REFERENCES I P. DAUDEL AND R. DAUDEL, Chemical Carcinogenesis and Molecular Biology, Interscience, J o h n Wiley, N e w York, 1966, p. 158. 2 C. HEIDELBERGER AND C-. R. DAVENPORT, Acta Unio Intern. Contra Cancrum, 17 (1961) 55. 3 p. BROOKES AND P. n . LAWLE¥, Rept. Brit. Empire Cancer Campaign, 4 ° (1962) 45. 4 P- BROOKES AND P. D. LAWLEY, Nature, 202 (1964) 781. 5 A. JACQtlIER AND P. DAUDEL, Compt. Rend., 258 (1964) 57756 P. L. GROVER AND P. SIMS, Biochem. J., IiO (1968) 159. 7 H. V. GELBOIN, Cancer Res., 29 (1969) 1272. 8 C. ~E. 1VIORREAL, T. L. DAS, K. ESKINS, C. L. KING AND J. DIENSTAG, Biochim. Biophys. Acta, 169 (1968) 224 . 9 R. S. UMANS, S. A. LESKO AND P. O. P. TS'O, Nature, 221 (1969) 763 . xo P. O. P. T s ' o a n d P. Lo, Proc. Natl. Acad. Sci. U.S., 51 (1964) 272. i i S. A. RAPAPORT AND P. O. P. TS'O, Biochemistry, 55 (1966) 381. 12 J. K. BALL, J. A. McCARTER AND M. F. SMITH, Biochim. Biophys. Acta, lO 3 (1965) 275. 13 J. P. RICHARDSON, in J. N. DAVlDSON AND W. E. COHN, Progress in Nucleic Acid Research and Molecular Biology, Vol. 9, A c a d e m i c Press, N e w York, 1969, p. 79. 14 O. H. IVERSEN AND A. EVENSEN, Acta t'athol. Microbiol. Scand. Suppl., 156 (1962). 15 J. A. MCCARTER AND H. QUASTLER, Biochim. Biophys. Acta, 55 (1962) 552. 16 M. CHAMBERLII~ AND P. BERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 81.
Biochim. Biophys. Acta, 238 (1971) 4 6 - 4 9