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The binding of polycyclic aromatic hydrocarbon diol-epoxides to DNA
Cancer Letters, 22 (1984) 95-98 Elsevier Scientific Publishers Ireland Ltd.
96
THE BINDING OF POLYCYCLIC AROMATIC HYDROCARBON DIOL-EPOXIDES TO DNA
PETER RLDLER and BARRY JENNINGS Electra-Optics 3PH (U.K.)
Group, Physics Department,
Brunel University, Uxbridge, Middlesex UB8
(Received 26 September 1983) (Accepted 15 December 1983)
SUMMARY
Diol-epoxide derivatives of polycyclic aromatic hydrocarbons are the ultimate carcinogenic forms of these compounds encountered in vivo. Using a novel electro-fluorescence apparatus, we report that, like the (+ )-anti-diolepoxide of benzo[a] pyrene (BP), the similar diol-epoxides of benzo[a] anthracen.e (BA) and 3-methylcholanthrene (3MC) also bind to the DNA helix at an inclination of approximately 50”. This compares with the essentially perpendicular, intercalative-type binding generally found with saturated aromatic Icompounds, such as the native hydrocarbons, and may indicate a systematic behaviour in the molecular associations precursive to carcinogenesis.
INTRODUCTION
Fluorescence is a two-fold optical property which involves the absorption of light by an active chemical group (fluorophore), followed by its subsequent re-emission at an increased wavelength. These processes occur along specific directional transition moments associated with the fluorophore structure. Recently we have developed a laser-based method [l] in which rapid changes are recorded in the polarised components of the relevant fluorescence, as dilute solutions of DNA-type complexes are subjected to the orientational torque of short duration, pulsed electric fields. From these data, any regular orientational ordering of the fluorophore transition moments relative to the DNA long axis can be determined. Hence the binding geometry of fluorescent chemical groups to DNA can be estimated. Certain polycyclic aromatic hydrocarbons, of which BP is the best known, are initiators of chemical carcinogenesis [2]. They are found in tars and combustion products and are strongly fluorescent. Recently, it has become clear that the animal host metabolises the native hydrocarbon into o 1984 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/84/$03.00 Published and Printed in Ireland
96
diol-epoxide derivatives [3], which are able to bind covalently to DNA [4]. The stereoisomeric forms of the diol-epoxides have differing carcinogenic potency [ 51, so that the unique features of the potent forms are sought in the quest for a fundamental understanding of their interaction mechanisms with DNA and of the subsequent molecular behaviour. In a recent publication [6], we used the electro-fluorescent method to show that, whereas the native form of BP binds to DNA with a geometric disposition similar to that of intercalative compounds, the in vivo ultimate carcinogen (?)-anti-benzo[a] pyrene-7,8dihydrodiol-9,10-epoxide (BPDE) binds to DNA in a regular manner and with an inclination of the long, in-plane molecular axis at approximately 50” to the helix and hence closely parallel to that of the helical grooves. BA, like BP, is a major constituent of coal tars and has been associated with cancer studies for many years [ 71. It can be converted to diol-epoxide forms, of which the (?)-anti-benzo[a]anthracene-3,4dihydrodiol-1,2epoxide (BADE) is thought to be the ultimate initiating carcinogen [8] . Despite the relative low potency of BA, the BADE is highly carcinogenic. The compound 3MC is a renowned carcinogen which has also been studied extensively [ 71. It also transforms to diol-epoxide derivatives, of which the (t)-anti-3-methylcholanthrene-9,lO-dihydrodiol-7,8-epoxide (SMCDE) is considered to be a carcinogenic initiator [8]. To our knowledge, no data exist on the configuration binding of either BADE or 3MCDE to DNA. Indeed, few methods exist with which such information can be obtained.
ifi
..I
::
iv
-I
‘if
Fig. 1. Transient changes induced in the vertically polarised component of the fluorescence with vertically polarised incident light. (i) for BADE/DNA complex; (ii) for 3MCDE/DNA complex. For comparison, data are also shown for (iii) BPDE/DNA complex, and (iv) for ethidium bromide/DNA system. In all cases excitation radiation of 351 nm and 364 nm wavelengths was used and detection was made for wavelengths exceeding 400 nm. The lowest trace is the applied electric pulse of 17 kV cm-’ and 0.5 ms duration. Time runs from left to right.
97 MATERLlLS
AND METHODS
Racemic BADE and 3MCDE were independently reacted with 6 X lo6 molecular mass DNA (ex calf thymus). The DNA was repeatedly extracted from aqueous solution with ether to remove any unreacted hydrocarbon including tetraols. Measurements were made on solutions at a DNA concentration of 10e4 g ml-’ with a carcinogen-to-base pair ratio as low as 1: 100. Vertical1.y polarised light at 351 nm and 364 nm wavelengths from an argonion laser was incident on the solutions and the horizontally (V, ) and vertically (V,,) polarised components of fluorescence detected at 90” to the incident light path and for wavelengths exceeding 400 nm. The transient changes (A V, and A V,) in these components were recorded as electric field pulses of up to 20 kV cm-’ amplitude (E) and 0.5 ms duration were applied :in a vertical direction across the samples. Typical responses are shown in Fig. 1 (i,ii) along with comparative data for the diol-epoxide of BP and ethidium bromide (a well-known intercalating compound). All data shown correspond to the high field condition [9] in which the transient amplitudes were invariant with E. DISCUSSION
From the data, 3 things are noted. First, as described elsewhere [l],intercalation is accompanied by a negative change in the component A V, for the geometry used here. This cannot be confused with the positive changes recorded for all 3 complexes involving the diol-epoxides. A very different binding igeometry is immediately apparent. Second, the BADE and 3MCDE complex.es give almost identical responses to each other and to the previously recorded measurements on BPDE. Third, quantitative values for the parameters were as follows: for BADE complex AV,/‘& = +0.06
AV,/V, = -0.10
V”lV, = 1.3
for 3MCDE complex A&&, = +0.08
AVh/Vh = -0.13
VJV,
= 1.4
From these, and using the theory described elsewhere [9], the inclinations \k and.9 ’ of the absorption and emission transition moments to the DNA long axis have the following identical values for both the BADE/DNA complex. and the 3MCDE/DNA complex; @ = 55 +4” and \k’ = 48 +-la. These should be compared with 52” and 45” already reported for the BPDE system. On the assumption that both moments lie within the plane of the anthracene rings and are along the long and short axis respectively [ 10,111 ; the inclination of the plane is indicated. It is evident that, although the native hydrocarbons BA and 3MC essen-
98
tially intercalate or have no interaction at all with DNA [12,13], the active diol-epoxide metabolites, like that of BP, incline at an angle of approximately 50” to the helix axis, which is similar to.the projected angle of the helical grooves. As the sites of interaction are as yet unknown for these systems, the significance of the observation is unclear. However, the observation that the 3 diol-epoxide derivatives BADE, 3MCDE and BPDE all bind with the same apparent geometry to DNA is thought to be of significance. Such a unique feature may hopefully lead to an enhanced understanding of the mechanisms of carcinogenic initiation at the molecular level. Extended studies are planned for other tumorigenic hydrocarbons. ACKNOWLEDGEMENTS
The authors thank Drs. P. Brookes and M.R. Osborne of the Institute of Cancer Research, Pollards Wood, for the provision of all samples. The M.R.C. is thanked for a research grant. REFERENCES 1 Jennings, B.R. and Ridler, P.J. (1977) Electrically induced fluorescence depolarisation of macromolecules. Chem. Phys. Lett., 45, 550-555. 2 Phillips, D.H. (1983) Fifty years of benzo(a)pyrene. Nature, 303,468-472. 3 Grover, P.I. and Sims, P. (1968) Enzyme-catalysed reaction of polycyclic hydrocarbons with deoxyribonucleic acid and protein in vitro. Biochem. J., 110, 159-160. 4 Heidelberger, C. and Davenport, G.R. (1961) Local functional components of carcinogenesis. Acta Un. Int. Caner., 17, 55-63. 5 Buening, M.K., Wislocki, P.G., Levin, W., Yagi, H., Thakker, D.R., Akagi, H., Koreeda, M., Jerina, D.M. and Conney, A.H. (1978) Tumorigenicity of the optical enantiomers of the diastereomeric benzo(a)pyrene 7,8-diol-9,10-epoxides in newborn mice: Exceptional activity of (+)-713,8a-dihydroxy-9or,lOa-epoxy-7,8,9,1O-tetrahydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. U.S.A., 75, 5358-5361. 6 Ridler, P.J. and Jennings, B.R. (1982) Electrically induced fluorescence as a method for studying benzo(a)pyrene binding to DNA. FEBS Lett., 139, 101-104. 7 Gelboin, H.V. and Ts’o, P.O.P. (Eds.) (1981) Polycyclic Aromatic Hydrocarbons and Cancer, Vols. 1, 2 and 3. Academic Press, New York. 8 Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydr\ocarbons: G.H.A. Clowes Memorial Lecture. Cancer Res., 42, 4875-4917. 9 Ridler, P.J. and Jennings, B.R. (1979) Electrically induced fluorescence changes from solutions of dye tagged polyribonucleotides. In: Electra-Optics dnd Dielectrics of Macromolecules and Colloids, pp. 99-107. Editor: B.R. Jennings. Plenun, New York. 10 Klevens, H. and Platt, J. (1963) Spectral resemblances of cata-condensed hydrocarbons. J. Chem. Phys., 17, 470-481. 11
12
13
Becker, R.S., Singh, I.S. and Jackson, E.A. (1963) Comprehensive spectroscopic investigation of polynuclear aromatic hydrocarbons. I Absorption spectra and state assignments for the tetracyclic hydrocarbons and their alkyl-substituted derivatives. J. Chem. Phys., 38, 9,2144-2171. Green, B. and McCarter, J.A. (1967) Polarised fluorescence of polycyclic hydrocarbons in aqueous DNA solutions. Effect of flow orientation. J. Mol. Biol., 23, 447456. Isenberg, I., Baird, S.L. and Bersohn, R. (1967) Interaction of polynucleotides with aromatic hydrocarbons. Biopolymers, 5, 477-482.
96
THE BINDING OF POLYCYCLIC AROMATIC HYDROCARBON DIOL-EPOXIDES TO DNA
PETER RLDLER and BARRY JENNINGS Electra-Optics 3PH (U.K.)
Group, Physics Department,
Brunel University, Uxbridge, Middlesex UB8
(Received 26 September 1983) (Accepted 15 December 1983)
SUMMARY
Diol-epoxide derivatives of polycyclic aromatic hydrocarbons are the ultimate carcinogenic forms of these compounds encountered in vivo. Using a novel electro-fluorescence apparatus, we report that, like the (+ )-anti-diolepoxide of benzo[a] pyrene (BP), the similar diol-epoxides of benzo[a] anthracen.e (BA) and 3-methylcholanthrene (3MC) also bind to the DNA helix at an inclination of approximately 50”. This compares with the essentially perpendicular, intercalative-type binding generally found with saturated aromatic Icompounds, such as the native hydrocarbons, and may indicate a systematic behaviour in the molecular associations precursive to carcinogenesis.
INTRODUCTION
Fluorescence is a two-fold optical property which involves the absorption of light by an active chemical group (fluorophore), followed by its subsequent re-emission at an increased wavelength. These processes occur along specific directional transition moments associated with the fluorophore structure. Recently we have developed a laser-based method [l] in which rapid changes are recorded in the polarised components of the relevant fluorescence, as dilute solutions of DNA-type complexes are subjected to the orientational torque of short duration, pulsed electric fields. From these data, any regular orientational ordering of the fluorophore transition moments relative to the DNA long axis can be determined. Hence the binding geometry of fluorescent chemical groups to DNA can be estimated. Certain polycyclic aromatic hydrocarbons, of which BP is the best known, are initiators of chemical carcinogenesis [2]. They are found in tars and combustion products and are strongly fluorescent. Recently, it has become clear that the animal host metabolises the native hydrocarbon into o 1984 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/84/$03.00 Published and Printed in Ireland
96
diol-epoxide derivatives [3], which are able to bind covalently to DNA [4]. The stereoisomeric forms of the diol-epoxides have differing carcinogenic potency [ 51, so that the unique features of the potent forms are sought in the quest for a fundamental understanding of their interaction mechanisms with DNA and of the subsequent molecular behaviour. In a recent publication [6], we used the electro-fluorescent method to show that, whereas the native form of BP binds to DNA with a geometric disposition similar to that of intercalative compounds, the in vivo ultimate carcinogen (?)-anti-benzo[a] pyrene-7,8dihydrodiol-9,10-epoxide (BPDE) binds to DNA in a regular manner and with an inclination of the long, in-plane molecular axis at approximately 50” to the helix and hence closely parallel to that of the helical grooves. BA, like BP, is a major constituent of coal tars and has been associated with cancer studies for many years [ 71. It can be converted to diol-epoxide forms, of which the (?)-anti-benzo[a]anthracene-3,4dihydrodiol-1,2epoxide (BADE) is thought to be the ultimate initiating carcinogen [8] . Despite the relative low potency of BA, the BADE is highly carcinogenic. The compound 3MC is a renowned carcinogen which has also been studied extensively [ 71. It also transforms to diol-epoxide derivatives, of which the (t)-anti-3-methylcholanthrene-9,lO-dihydrodiol-7,8-epoxide (SMCDE) is considered to be a carcinogenic initiator [8]. To our knowledge, no data exist on the configuration binding of either BADE or 3MCDE to DNA. Indeed, few methods exist with which such information can be obtained.
ifi
..I
::
iv
-I
‘if
Fig. 1. Transient changes induced in the vertically polarised component of the fluorescence with vertically polarised incident light. (i) for BADE/DNA complex; (ii) for 3MCDE/DNA complex. For comparison, data are also shown for (iii) BPDE/DNA complex, and (iv) for ethidium bromide/DNA system. In all cases excitation radiation of 351 nm and 364 nm wavelengths was used and detection was made for wavelengths exceeding 400 nm. The lowest trace is the applied electric pulse of 17 kV cm-’ and 0.5 ms duration. Time runs from left to right.
97 MATERLlLS
AND METHODS
Racemic BADE and 3MCDE were independently reacted with 6 X lo6 molecular mass DNA (ex calf thymus). The DNA was repeatedly extracted from aqueous solution with ether to remove any unreacted hydrocarbon including tetraols. Measurements were made on solutions at a DNA concentration of 10e4 g ml-’ with a carcinogen-to-base pair ratio as low as 1: 100. Vertical1.y polarised light at 351 nm and 364 nm wavelengths from an argonion laser was incident on the solutions and the horizontally (V, ) and vertically (V,,) polarised components of fluorescence detected at 90” to the incident light path and for wavelengths exceeding 400 nm. The transient changes (A V, and A V,) in these components were recorded as electric field pulses of up to 20 kV cm-’ amplitude (E) and 0.5 ms duration were applied :in a vertical direction across the samples. Typical responses are shown in Fig. 1 (i,ii) along with comparative data for the diol-epoxide of BP and ethidium bromide (a well-known intercalating compound). All data shown correspond to the high field condition [9] in which the transient amplitudes were invariant with E. DISCUSSION
From the data, 3 things are noted. First, as described elsewhere [l],intercalation is accompanied by a negative change in the component A V, for the geometry used here. This cannot be confused with the positive changes recorded for all 3 complexes involving the diol-epoxides. A very different binding igeometry is immediately apparent. Second, the BADE and 3MCDE complex.es give almost identical responses to each other and to the previously recorded measurements on BPDE. Third, quantitative values for the parameters were as follows: for BADE complex AV,/‘& = +0.06
AV,/V, = -0.10
V”lV, = 1.3
for 3MCDE complex A&&, = +0.08
AVh/Vh = -0.13
VJV,
= 1.4
From these, and using the theory described elsewhere [9], the inclinations \k and.9 ’ of the absorption and emission transition moments to the DNA long axis have the following identical values for both the BADE/DNA complex. and the 3MCDE/DNA complex; @ = 55 +4” and \k’ = 48 +-la. These should be compared with 52” and 45” already reported for the BPDE system. On the assumption that both moments lie within the plane of the anthracene rings and are along the long and short axis respectively [ 10,111 ; the inclination of the plane is indicated. It is evident that, although the native hydrocarbons BA and 3MC essen-
98
tially intercalate or have no interaction at all with DNA [12,13], the active diol-epoxide metabolites, like that of BP, incline at an angle of approximately 50” to the helix axis, which is similar to.the projected angle of the helical grooves. As the sites of interaction are as yet unknown for these systems, the significance of the observation is unclear. However, the observation that the 3 diol-epoxide derivatives BADE, 3MCDE and BPDE all bind with the same apparent geometry to DNA is thought to be of significance. Such a unique feature may hopefully lead to an enhanced understanding of the mechanisms of carcinogenic initiation at the molecular level. Extended studies are planned for other tumorigenic hydrocarbons. ACKNOWLEDGEMENTS
The authors thank Drs. P. Brookes and M.R. Osborne of the Institute of Cancer Research, Pollards Wood, for the provision of all samples. The M.R.C. is thanked for a research grant. REFERENCES 1 Jennings, B.R. and Ridler, P.J. (1977) Electrically induced fluorescence depolarisation of macromolecules. Chem. Phys. Lett., 45, 550-555. 2 Phillips, D.H. (1983) Fifty years of benzo(a)pyrene. Nature, 303,468-472. 3 Grover, P.I. and Sims, P. (1968) Enzyme-catalysed reaction of polycyclic hydrocarbons with deoxyribonucleic acid and protein in vitro. Biochem. J., 110, 159-160. 4 Heidelberger, C. and Davenport, G.R. (1961) Local functional components of carcinogenesis. Acta Un. Int. Caner., 17, 55-63. 5 Buening, M.K., Wislocki, P.G., Levin, W., Yagi, H., Thakker, D.R., Akagi, H., Koreeda, M., Jerina, D.M. and Conney, A.H. (1978) Tumorigenicity of the optical enantiomers of the diastereomeric benzo(a)pyrene 7,8-diol-9,10-epoxides in newborn mice: Exceptional activity of (+)-713,8a-dihydroxy-9or,lOa-epoxy-7,8,9,1O-tetrahydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. U.S.A., 75, 5358-5361. 6 Ridler, P.J. and Jennings, B.R. (1982) Electrically induced fluorescence as a method for studying benzo(a)pyrene binding to DNA. FEBS Lett., 139, 101-104. 7 Gelboin, H.V. and Ts’o, P.O.P. (Eds.) (1981) Polycyclic Aromatic Hydrocarbons and Cancer, Vols. 1, 2 and 3. Academic Press, New York. 8 Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydr\ocarbons: G.H.A. Clowes Memorial Lecture. Cancer Res., 42, 4875-4917. 9 Ridler, P.J. and Jennings, B.R. (1979) Electrically induced fluorescence changes from solutions of dye tagged polyribonucleotides. In: Electra-Optics dnd Dielectrics of Macromolecules and Colloids, pp. 99-107. Editor: B.R. Jennings. Plenun, New York. 10 Klevens, H. and Platt, J. (1963) Spectral resemblances of cata-condensed hydrocarbons. J. Chem. Phys., 17, 470-481. 11
12
13
Becker, R.S., Singh, I.S. and Jackson, E.A. (1963) Comprehensive spectroscopic investigation of polynuclear aromatic hydrocarbons. I Absorption spectra and state assignments for the tetracyclic hydrocarbons and their alkyl-substituted derivatives. J. Chem. Phys., 38, 9,2144-2171. Green, B. and McCarter, J.A. (1967) Polarised fluorescence of polycyclic hydrocarbons in aqueous DNA solutions. Effect of flow orientation. J. Mol. Biol., 23, 447456. Isenberg, I., Baird, S.L. and Bersohn, R. (1967) Interaction of polynucleotides with aromatic hydrocarbons. Biopolymers, 5, 477-482.