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The binding of benzo[α] pyrene to DNA does not involve substitution at the 6-position
Chem.-Biol. Interactions, 12 (1976) 425-428 0 Elsevier Scientific Publishing Company, Amsterdam
Short communication --.--.-
..___--
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- Printed in The Netherlands
l--l_-ll-
THE BINDING OF BENZO[a] PYRENE TO DNA DOES NOT INVOLVE SUBSTITUTION AT THE 6-POSITION
H.W.S. KING, M.H. THOMPSON, MR. OSBORNE, and P. BROOKES l *
R.G. HARVEY
l
Chemical Carcinogenesis Division. Pollards Wood Research Station, Institute of Cancer Research, Nightingales Lane, Chalfont St. Giles, Buckinghamshire HP8 4SP (Great Britain) and * Ben May Laboratory for Cancer Research, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received October 27th, 1975) (Accepted November Filth, 1975)
As reported earlier [l] the retention of tritium when [ jH] [ l*C] benzo[a] pyrene was bound to DNA as a consequence of metabolism by cells in culture accorded with the view of Sims et al. [2] that a diol-epoxide intermediate was responsible for the in vivo reaction of benzo[a]pyrene (BP) with DNA. In the earlier studies [l] randomly tritiated BP was used and since 6-hydroxybenzo[a] pyrene (6-OH-BP) is not isolated as a metabolite of BP [3], the tritium loss which would be expected for a DNA binding reaction involving the 6-position could not be determined. The specific involvement of the 6-position in the activation of BP was proposed by Nagata et al. [4] and supported by the studies of T’so et al. j5) who proposed the formation of a 6-phenoxy free radical intermediate derived perhaps from 6-OH-BP. An alternative mechanism of activation which also involved substitution at the 6-position was suggested by Flesher and Sydnor [6] who proposed the metabolic formation of 6-hydroxymethylbenzo[a] pyrene. The possibility of obtaining BP labelled specifically in the 6-position with tritium and the methods described previously [l] using double-lab&d BP, have enabled us to assess the contribution made by metabolism at the 6position of BP to the DNA binding occurring both in cells in culture and mediated by rat liver microsomes in vitro.
* To whom reprint requests should be sent. 6.Br-BP, Abbreviations: BP, benzo[a]pyrene; hydroxybenzo[a]-pyrene. l
6-bromobenzo[a]
pyrene;
6-GH-BP,
6-
425
MATERIALS
AND METHODS
[6-jH] BP was prepared at the Radiochemical Centre, Amersham, Bucks., lEngland, from 6-bromobenzo[a] pyrene (6-Br-BP) by metal catalysed halogen$xitium replacement according to the method described by Harvey and Fu [7]. The 6-Br-BP was supplied by us and was synthesised as described by :Dewhurst and Kitchen [8]. The [6-“H] BP had a specific radioactivity of 36 Ci/mmole and 95% of the Lritium was in the 6-position as determined by tritium n.m.r. studies (J. Elvidge, University of Surrey and A.E. Evans, Radiochemical Centre, personal communication). Other materials and the methods for the metabolism and binding of [ 6-“H] [‘“C] BP to DNA and subsequent degradation and product isolation were essentially as described previously [ 11, or as stated in Tables I and II. RESULTS AND DISCUSSION
Thin-layer chromatography of the metabolites produced by rat-liver microsomal metabolism of [6-‘H] [‘*Cl BP gave the results indicated in Table I. The products, located by their colour or fluorescence under UV-light were identified by spectrophotometric and TLC comparison with marker compounds. Elution of individual products into methanol and the determination of their tritium to carbon-14 ratio in comparison with that of the recovered unmetabolised [ 6-3H] [‘*Cl BP enabled the loss of tritium accompanying formation of each1 metabolite to be assessed (Table I). Only in the
TABLE I THE TRITIUM RELATIVE TO CARBON-14 CONTENT OF BENZO[a]PYRENE ITS MICROSOMAL METABOLITES SEPARATED BY TLC
AND
The data from microsomal incubation and subsequent TLC are given. The chromatography employed silica gel plates which were developed initially with hexane:benzene (14:l) followed by benzene:methanol (14:l). The ratio of [3H] : [‘*Cl in the eluted products was determined at a counting efficiency of 41% for [ JH ] and 61% for [‘*Cl. .-_ Nature of product
Unknown BP-9,10-diol BP-7,&diol BP-4,5-diol 3-OH-BP (plus trace of g-OH-BP) [3] Quinone I Quinone II BP
426
RF
Appearance in visible or UV light
0.06 0.22 0.33 0.39
Grey (UV) Grey (UV) Violet (UV) Grey (UV)
0.55 0.72 0.76 0.89
g;t
WV)
Yellow Violet (UV)
-_Mean [SH] : [‘*Cl ratio
[“H] Retention relative to BP ---
8.6 13.5 12.2 12.5
63% 99% 90% 93%
12.5 1.3 1.0 13.6
92% 10% 7% 100%
TABLE II THE TRITIUM RELATIVE TO r; ARBCN-I DEOXYRIBONUCLEOSIDE PRODUCTS DERIVED FRO BP BOUND IN MOUSE CELLS [9 1 OR CATAL:SED SOMAL SYSTEM [ 10 ]
BY A R
Products assayed
Lives microsomal experiment [3H] : [‘4C]BPaddedtomicrosomal incubation system Product eluting from LH20 Sephadex column at 570 ml elution volume (i.e. Peak A of ref. 10) 13.9
98%
to embryo
12.6
106%
Product eluting from LH20 Sephadex at 570 ml (i.e. “in vivo product”, of ref. 9)
12.0
95
Product eluting from LH20 Sephadex column at 780 ml (i.e. Peak D, of ref. 10)
DNA from mouse embryo cells [3H] : [iW]BPadded cells in culture
-
~_____.
__ -._^_ -_
case of the quinones and unknown material near the origin was the mourn loss greater than 10%. It is of interest that metabolism at the v of the BP ring system leading to the severaI phenol and dihy olites identified, does not result in any significant labilisation of the tritium in the 6-position. Enzymatic degradation and fractionation on LII20 isolated from mouse embryo cells exposed to r3H] [‘“C] a single product as reported previously [ 9,1] . The [‘WI : so called “in vivo product’* showed it to have retained 9 the starting hydrocarbon (Table II). Similarly DNA with r3H] [ 14C]BP bound du in the presence of added native DNA was de LH20 Sephadex. The elution profile showed two m ly reported [lo]. The tritium to carbon-14 were determined and indicated negligible loss of tritium The present data rule out the possibility that the either in cells or as mediated by microsomes in vitro involv the 6-position. It also confirms the previous findin tion does not precede DNA reaction.
ACKNOWLEDGEMENTS
We would like to express our thanks to Jacqueline Amos for excellent technical assistance throughout the course of this work. The work was supported by N.I.H. (U.S.A.) contracts number NOl-CP33367 and 33385 (RCH) and in part by grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign, also (RGH) Grant E-132 from the American Cancer Society. REFERENCES M.R. Osborne, M.H. Thompson, H.W.S. King and P. Brookes, Retention of tritium during the binding of tritiated benzofalpyrene to DNA, Int. J. Cancer, 16 (1975) 659. 2 P. Sims, P.L. Grover, A, Swaisland, K. Pal and A. Hewer, Metabolic activation of benzo[a]pyrene proceeds by a diol epoxide, Nature, 252 (1974) 326. 3 J.K. Selkirk, R.C. Gray, P.P. Roller and H.V. Gelboin, High-pressure liquid chromatographic analysis of benzo[o]pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzo~avone and 1,2~poxy-3,3,3-trichloropropane~ Cancer Res., 34 (1974) 3474. 4 C. Nagata, Y. Tagashira and M. Kodama, Metabolic activation of benzo[o lpyrene: Significance of the free radical, in P.O.P. T’so and J.A. DiPaolo (Eds.), Chemical Carcinogenesis, Part A. Marcel Dekker, New York, 1974, pp. 87-112. 5 P.O.P. T’so, W.J. Caspary, B.I. Cohen, J.C. Leavitt, S.A. Lesko, R. J. Lorentzen and L.M. Schechtman in P.O.P. T’so and J.D. DiPaoio (Eds.), Chemical Carcinogenesis, Part A, Marcel Dekker, New York, 1974, pp. 113-148. 6 J.W. Fiesher and K.L. Sydnor, Possible role of 6-hydroxymeth~~benao~a Jpyrene as a proximate carcinogen of benzo[a]pyrene and 6-methylbenzo[of pyrenc, Int. J. Cancer, 11 (1973) 433. 7 R.G, Harvey and P.P. Fu, Synthesis of high specific activity benlzo[a]pyrene and its K-region oxidised derivatives, J. Label Compounds, (1976) (in press). E: F. Dewhurst and D.A. Kitchen, Synthesis and properties of 6-substituted benzo[o]pyrene derivatives, J. Chem. Sot., Perkin I, (1972) 710. 9 W.M. Baird, R.G. Harvey and P. Brookes, Comparison of the cellular DNA-bound producis of benzo[o]pyrene with the products formed by the reaction of benzo[o]pyrene-4,5-oxide with DNA, Cancer Res., 35 (1975) 54. 10 H.W.S. King, M.H. Thompson and P. Brookes, The benzo[a]pyrene deoxyribonucleoside products isolated from DNA after metabolism of benzo[a ] pyrene by rat liver microsomes in the presence of DNA, Cancer Res., 35 (1975) 1263. 1
428
Short communication --.--.-
..___--
-
- Printed in The Netherlands
l--l_-ll-
THE BINDING OF BENZO[a] PYRENE TO DNA DOES NOT INVOLVE SUBSTITUTION AT THE 6-POSITION
H.W.S. KING, M.H. THOMPSON, MR. OSBORNE, and P. BROOKES l *
R.G. HARVEY
l
Chemical Carcinogenesis Division. Pollards Wood Research Station, Institute of Cancer Research, Nightingales Lane, Chalfont St. Giles, Buckinghamshire HP8 4SP (Great Britain) and * Ben May Laboratory for Cancer Research, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received October 27th, 1975) (Accepted November Filth, 1975)
As reported earlier [l] the retention of tritium when [ jH] [ l*C] benzo[a] pyrene was bound to DNA as a consequence of metabolism by cells in culture accorded with the view of Sims et al. [2] that a diol-epoxide intermediate was responsible for the in vivo reaction of benzo[a]pyrene (BP) with DNA. In the earlier studies [l] randomly tritiated BP was used and since 6-hydroxybenzo[a] pyrene (6-OH-BP) is not isolated as a metabolite of BP [3], the tritium loss which would be expected for a DNA binding reaction involving the 6-position could not be determined. The specific involvement of the 6-position in the activation of BP was proposed by Nagata et al. [4] and supported by the studies of T’so et al. j5) who proposed the formation of a 6-phenoxy free radical intermediate derived perhaps from 6-OH-BP. An alternative mechanism of activation which also involved substitution at the 6-position was suggested by Flesher and Sydnor [6] who proposed the metabolic formation of 6-hydroxymethylbenzo[a] pyrene. The possibility of obtaining BP labelled specifically in the 6-position with tritium and the methods described previously [l] using double-lab&d BP, have enabled us to assess the contribution made by metabolism at the 6position of BP to the DNA binding occurring both in cells in culture and mediated by rat liver microsomes in vitro.
* To whom reprint requests should be sent. 6.Br-BP, Abbreviations: BP, benzo[a]pyrene; hydroxybenzo[a]-pyrene. l
6-bromobenzo[a]
pyrene;
6-GH-BP,
6-
425
MATERIALS
AND METHODS
[6-jH] BP was prepared at the Radiochemical Centre, Amersham, Bucks., lEngland, from 6-bromobenzo[a] pyrene (6-Br-BP) by metal catalysed halogen$xitium replacement according to the method described by Harvey and Fu [7]. The 6-Br-BP was supplied by us and was synthesised as described by :Dewhurst and Kitchen [8]. The [6-“H] BP had a specific radioactivity of 36 Ci/mmole and 95% of the Lritium was in the 6-position as determined by tritium n.m.r. studies (J. Elvidge, University of Surrey and A.E. Evans, Radiochemical Centre, personal communication). Other materials and the methods for the metabolism and binding of [ 6-“H] [‘“C] BP to DNA and subsequent degradation and product isolation were essentially as described previously [ 11, or as stated in Tables I and II. RESULTS AND DISCUSSION
Thin-layer chromatography of the metabolites produced by rat-liver microsomal metabolism of [6-‘H] [‘*Cl BP gave the results indicated in Table I. The products, located by their colour or fluorescence under UV-light were identified by spectrophotometric and TLC comparison with marker compounds. Elution of individual products into methanol and the determination of their tritium to carbon-14 ratio in comparison with that of the recovered unmetabolised [ 6-3H] [‘*Cl BP enabled the loss of tritium accompanying formation of each1 metabolite to be assessed (Table I). Only in the
TABLE I THE TRITIUM RELATIVE TO CARBON-14 CONTENT OF BENZO[a]PYRENE ITS MICROSOMAL METABOLITES SEPARATED BY TLC
AND
The data from microsomal incubation and subsequent TLC are given. The chromatography employed silica gel plates which were developed initially with hexane:benzene (14:l) followed by benzene:methanol (14:l). The ratio of [3H] : [‘*Cl in the eluted products was determined at a counting efficiency of 41% for [ JH ] and 61% for [‘*Cl. .-_ Nature of product
Unknown BP-9,10-diol BP-7,&diol BP-4,5-diol 3-OH-BP (plus trace of g-OH-BP) [3] Quinone I Quinone II BP
426
RF
Appearance in visible or UV light
0.06 0.22 0.33 0.39
Grey (UV) Grey (UV) Violet (UV) Grey (UV)
0.55 0.72 0.76 0.89
g;t
WV)
Yellow Violet (UV)
-_Mean [SH] : [‘*Cl ratio
[“H] Retention relative to BP ---
8.6 13.5 12.2 12.5
63% 99% 90% 93%
12.5 1.3 1.0 13.6
92% 10% 7% 100%
TABLE II THE TRITIUM RELATIVE TO r; ARBCN-I DEOXYRIBONUCLEOSIDE PRODUCTS DERIVED FRO BP BOUND IN MOUSE CELLS [9 1 OR CATAL:SED SOMAL SYSTEM [ 10 ]
BY A R
Products assayed
Lives microsomal experiment [3H] : [‘4C]BPaddedtomicrosomal incubation system Product eluting from LH20 Sephadex column at 570 ml elution volume (i.e. Peak A of ref. 10) 13.9
98%
to embryo
12.6
106%
Product eluting from LH20 Sephadex at 570 ml (i.e. “in vivo product”, of ref. 9)
12.0
95
Product eluting from LH20 Sephadex column at 780 ml (i.e. Peak D, of ref. 10)
DNA from mouse embryo cells [3H] : [iW]BPadded cells in culture
-
~_____.
__ -._^_ -_
case of the quinones and unknown material near the origin was the mourn loss greater than 10%. It is of interest that metabolism at the v of the BP ring system leading to the severaI phenol and dihy olites identified, does not result in any significant labilisation of the tritium in the 6-position. Enzymatic degradation and fractionation on LII20 isolated from mouse embryo cells exposed to r3H] [‘“C] a single product as reported previously [ 9,1] . The [‘WI : so called “in vivo product’* showed it to have retained 9 the starting hydrocarbon (Table II). Similarly DNA with r3H] [ 14C]BP bound du in the presence of added native DNA was de LH20 Sephadex. The elution profile showed two m ly reported [lo]. The tritium to carbon-14 were determined and indicated negligible loss of tritium The present data rule out the possibility that the either in cells or as mediated by microsomes in vitro involv the 6-position. It also confirms the previous findin tion does not precede DNA reaction.
ACKNOWLEDGEMENTS
We would like to express our thanks to Jacqueline Amos for excellent technical assistance throughout the course of this work. The work was supported by N.I.H. (U.S.A.) contracts number NOl-CP33367 and 33385 (RCH) and in part by grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign, also (RGH) Grant E-132 from the American Cancer Society. REFERENCES M.R. Osborne, M.H. Thompson, H.W.S. King and P. Brookes, Retention of tritium during the binding of tritiated benzofalpyrene to DNA, Int. J. Cancer, 16 (1975) 659. 2 P. Sims, P.L. Grover, A, Swaisland, K. Pal and A. Hewer, Metabolic activation of benzo[a]pyrene proceeds by a diol epoxide, Nature, 252 (1974) 326. 3 J.K. Selkirk, R.C. Gray, P.P. Roller and H.V. Gelboin, High-pressure liquid chromatographic analysis of benzo[o]pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzo~avone and 1,2~poxy-3,3,3-trichloropropane~ Cancer Res., 34 (1974) 3474. 4 C. Nagata, Y. Tagashira and M. Kodama, Metabolic activation of benzo[o lpyrene: Significance of the free radical, in P.O.P. T’so and J.A. DiPaolo (Eds.), Chemical Carcinogenesis, Part A. Marcel Dekker, New York, 1974, pp. 87-112. 5 P.O.P. T’so, W.J. Caspary, B.I. Cohen, J.C. Leavitt, S.A. Lesko, R. J. Lorentzen and L.M. Schechtman in P.O.P. T’so and J.D. DiPaoio (Eds.), Chemical Carcinogenesis, Part A, Marcel Dekker, New York, 1974, pp. 113-148. 6 J.W. Fiesher and K.L. Sydnor, Possible role of 6-hydroxymeth~~benao~a Jpyrene as a proximate carcinogen of benzo[a]pyrene and 6-methylbenzo[of pyrenc, Int. J. Cancer, 11 (1973) 433. 7 R.G, Harvey and P.P. Fu, Synthesis of high specific activity benlzo[a]pyrene and its K-region oxidised derivatives, J. Label Compounds, (1976) (in press). E: F. Dewhurst and D.A. Kitchen, Synthesis and properties of 6-substituted benzo[o]pyrene derivatives, J. Chem. Sot., Perkin I, (1972) 710. 9 W.M. Baird, R.G. Harvey and P. Brookes, Comparison of the cellular DNA-bound producis of benzo[o]pyrene with the products formed by the reaction of benzo[o]pyrene-4,5-oxide with DNA, Cancer Res., 35 (1975) 54. 10 H.W.S. King, M.H. Thompson and P. Brookes, The benzo[a]pyrene deoxyribonucleoside products isolated from DNA after metabolism of benzo[a ] pyrene by rat liver microsomes in the presence of DNA, Cancer Res., 35 (1975) 1263. 1
428