Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 979-983, March 1977 Biochemistry
Excision repair of benzo[a]pyrene-deoxyguanosine adducts in baby hamster kidney 21/C13 cells and in secondary mouse embryo fibroblasts C57BL/6J (benzo[aJpyrene-DNA adducts)
KUNIO SHINOHARA AND PETER A. CERUTTI Department of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida, Gainesville, Fla. 32610
Communicated by Bernhard Witkop, January 3, 1977
The formation and excision of benzo[alpyrene-deoxyguanosine adducts in metabolizing baby hamster kidney cells (21/C13) and secondary mouse embryo fibroblasts (C57BL/6J) was investigated. Both diastereomeric adducts, N2--(10-$7ft,8a,9a-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[aJpyrenejyl)deoxyguanosine and N2(10-f7ft,8a,9#-trihydroxy7,8,9,10-tetrahydrobenzo[alpyrenejyl)deoxyguanosine, were detected in both cell lines and both cell lines were capable of excising these lesions, albeit with low efficiency. There is much support for the notion that damage to the chromosomal DNA is a necessary initial step in physical and chemical carcinogenesis. Many carcinogens have been shown unambiguously to be DNA damaging agents (1-3). Most cells have evolved elaborate mechanisms for the processing of DNA damage. In general, prereplication excision repair-i.e., repair completed before the replication fork reaches the damaged residues8appears to accomplish complete restitution of the structure and activities of the DNA in prokaryotic and eukaryotic cells (4). Biological effects of DNA damaging agents such as cell death, mutation, and malignant transformation appear to be consequences of DNA replication on templates containing unexcised lesions. Therefore, the capacity of a cell to remove damaged residues from DNA by prereplication repair is expected to affect the magnitude of the biological effects (5, 6). The structural features of unexcised lesions may in turn affect the lethal, mutagenic, and carcinogenic efficiencies of the damaging agent (e.g., see refs. 7 and 8). Benzo[a]pyrene (B[a]P) is an ubiquitous pollutant of our environment and is suspected to be carcinogenic in man (9). It has been shown that covalent adducts are formed between B[a]P and DNA in metabolizing mammalian cells, and there is strong evidence that the major products formed are the stereoisomeric N2-(10-1713,8a,9a- or 9f-trihydroxy-7,8,9,10tetrahydrobenzo[a Jpyrenelyl)deoxyguanosines (dGua-B[a ]P I or dGua-B[a]P II, respectively) (10-14). The active B[a]P metabolites reacting with the exocyclic amino group of deoxyguanosine during formation of these adducts are the diastereomeric (+)7f.,8a-dihydroxy-9a,10a or 9,B,10#-epoxy7,8,9,10-tetrahydrobenzo[a]pyrenes (13-20) (B[a]P-diol-expoxides I or II, respectively). It has recently been shown that the B[a ]P-diol-epoxides are exceptionally potent mutagens in Salmonella and in V79 hamster cells (21-25). According to the concepts discussed above, it is expected that a determining Abbreviations: B[alP, benzo[a]pyrene; dGua-B[a]P I, N2-(10J7f,8a,9a-trihydroxy-7, 8,9, 10-tetrahydrobenzo[a pyrenelyl)deoxyguanosine; dGua-B[a]P II, N2-(10-t7B,8a,9fl-trihydroxy-7,8,9,1O-tetrahydrobenzo[a pyrenejyl)deoxyguanosine; B[a]P-diol-epoxide I, (+)7fB,8a-dihydroxy-9a,10a-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-diol-epoxide II, (b)7j6,8a-dihydroxy-9fl,10B-epoxy7,8,9,10-tetrahydrobenzo[ajpyrene; BHK, baby hamster kidney cells; ABSTRACT
MEF, mouse embryo fibroblasts.
979
factor for the biological effects of B[a]P, besides quality and quantity of metabolic activation, is the capacity of a cell to remove the B[a ]P-induced DNA lesions by prereplication excision repair. We are reporting our studies of the removal of dGuaB[a ]P adducts, during post-treatment incubation, from the DNA of baby hamster kidney cells (21/C13) (BHK) and secondary mouse embryo cells (C57BL/6J) (MEF). It was found that both cell lines were able to remove these lesions from their DNA, albeit slowly. The relative persistence of dGua-B[a]P lesions may be a factor in the mutagenic activity of the B[a] P-diol-epoxides. MATERIALS AND METHODS Chemicals. Generally labeled [3H]benzo[a]pyrene (20 Ci/ mmol) was purchased from Amersham/Searle, Arlington Heights, Ill., and dissolved in dimethyl sulfoxide at a concentration of 25 mCi/3.2 ml. [2-14C]Thymidine (61 mCi/mmol) was supplied by the same company. Cell Cultures. BHK were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 10% tryptose phosphate broth solution (29.5 g/liter) under 10% CO2 at 100% humidity. Primary monolayer cultures of MEF were prepared from 16- to 18-day-old C57BL/6J mouse embryos; 5 X 106 cells were inoculated in T150 Corning plastic culture flasks and grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum under 5% CO2 at 100% humidity. Three days later, the cells were harvested and replated at 1 X 106 cells per T150 flask for the experiments. Benzo[ajpyrene Treatment and Post-Treatment Incubation. Approximately 3 X 105 BHK were inoculated into T150 culture flasks, 30 ml of medium was added, and the cultures were incubated for 30-36 hr. Under our growth conditions, BHK have an approximate generation time of 12 hr. Radioactive thymidine (6 nCi of [2-14C]thymidine), cold thymidine (0.03 ,tmol), and cold deoxycytidine (0.03 Mumol) were then added to each culture flask and the same additions were repeated four times at 12-hr intervals. Together with the second addition of [14C]thymidine, 0.1 ml of [3H]B[a]P stock solution in dimethyl sulfoxide was added (final concentration, 0.33 ,ug of B[a ]P per ml growth medium). The cells were washed with fresh prewarmed medium containing 1 ,uM cold thymidine 48 hr after the addition of [3H]B[a]P. The protocol for labeling of BHK with [2-14C]thymidine and treatment with [3H]B[a]P is further described in Table 1. Under these conditions the cultures were almost confluent, the cellular DNA was uniformly labeled with [14C]thymidine, and B[a]P was almost completely metabolized to water-soluble derivatives. The cells were harvested by trypsinization, washed twice, and replated into T150 flasks at I to 2 X 106 cells per flask, and growth was continued up to 72 hr in medium containing 1 MM cold thymidine. After
Biochemistry: Shinohara and Cerutti
980
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Protocol for labeling of BHK with [2- lC ] thymidine and treatment with [3H ]benzo[a ]pyrene Time, hr
Step
0 36 48
5 x 10 BHK in 30 ml of growth medium Add: [4C ]dThd; cold dThd; cold dCyd Add: [4C ]dThd; cold dThd; cold dCyd;
Same as 36 hr Same as 36 hr Same as 36 hr Cells washed, harvested, and replated for post-treatment incubation
post-treatment incubation, the cells were harvested and washed several times with Hanks' balanced salt solution. Analogous procedures were used for MEF except that the labeling protocol was adjusted to the generation time of approximately 24 hr. At the outset of the experiment, the cells were in their second passage. [2-'4C]thymidine (20 nCi), cold thymidine (0.03 ,umol), and cold deoxycytidine (0.03,umol) were added three times in 24-hr intervals. Radioactive B[a 1P (final concentration, 0.33 tg/ml of growth medium) was added with the second addition of ['4C]thymidine. The B[a]P was almost completely metabolized to water-soluble derivatives within 48 hr. The cells were harvested, washed, replated, and subjected to -post-treatment incubation as described for BHK. DNA Extraction and Enzymatic Hydrolysis. The methods for DNA extraction were those of Diamond et al. (26) and Duncan et al. (27), and the conditions for the digestion of the DNA to nucleosides were those described by Baird and Brookes B
1500 - A
BHK -
I
2500 -2500 n
5o
I
ON
[3HJB[a]P
60 72 84 96
E 1000
BHK
u
I ___ I_
a II p . 20 0
3
h-
-
20
40
60
80
0
300
I.
E
-50 3
I FRACTION NUMBER
FIG. 1. Sephadex LH-20 chromatograms of DNA--digests from BHK (Upper) and secondary MEF (Lower) that had be-en exposedto see 1[4C]thymidine and [3H]B[a]P (for experimental prn Materials and Methods). Peaks I and II in part B of ea ch chromatogram correspond to the deoxyguanosine-benzo[a]pyr -ene adducts, dGua-B[a]P I and dGua-B[a]P II. The horizontal bar, sgive the position of elution of 4-p-nitrobenzylpyridine which was added as absorbancy marker to each sample.
,cedures,
0.
o20 10,
I
B
40
60 ~~~~~~I
80
MEF
0.4 0.3-
0.2-
0.
20 60 40 FRACTION NUMBER
80
FIG. 2. Removal of dGua-BlaJP I and dGua-B[a]P II from the DNA of BHK and secondary MEF during post-treatment incubation. Part B of Sephadex LH-20 chromatograms of DNA digests from BHK (Upper) and secondary MEF (Lower) that had been exposed to [14C]thymidine and [3H]B[a]P and then incubated in fresh medium for 0 and 72 hr. The ratios of the 3H radioactivity contained in each fraction to total 14C radioactivity eluted from the column are plotted (note that all 14C radioactivity is contained in part A of the chromatogram as is shown in Fig. 1). The horizontal bars give the position of elution of 4-p-nitrobenzylpyridine which was added as absorbancy marker to each sample.
(28). The following minor modifications were made: (i) the extracted DNA was precipitated once with ethanol; (ii) double the amounts of hydrolytic enzymes were used for DNA digestion; and (iii) phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) was added in two installments at the beginning and after 24 hr of the time allowed for digestion. Under these conditions, the 14C label was renderedcompletely soluble in 10% (wt/vol) trichloroacetic acid. The 3H label was not completely solubilized. This is attributed to the limited solubility of benzo[a]pyrene-substituted nucleosides in trichloroacetic acid. No evidence for the formation of oligonucleotide limit digests was obtained from the Sephadex LH-20 chromatograms (see below). Sephadex LH-20 Chromatography. A small amount of 4p-nitrobenzylpyridine was added to the DNA digests as 'absorbance marker and the samples were applied to 0.9 X 24 cm Sephadex LH-20 columns. The columns were first eluted with 30% (vol/vol) aqueous methanol and twenty-four 1.4-ml fractions were collected. The columns were further eluted with a linear gradient of 30% aqueous methanol/sodium borate to 80% aqueous methanol/sodium borate. The starting solvent was prepared by adding 26 ml of methanol to 61 ml of 0.05 M sodium borate at pH 8.7, and the final solvent contained 80 ml of methanol and 20 ml of 0.05 M sodium borate at pH 8.7. Seventy-six 1.4-ml fractions were collected and their radioactivity was counted in a Beckman model LS 233 scintillation system after the addition of 14 ml of Aquasol to each fraction.
Recovery of 14C radioactivity from the columns was always
complete; the recovery of 3H radioactivity was 90-100%.
Biochemistry: Shinohara, and Cerutti
Proc. Natl. Acad. Sci. USA 74 (1977)
981
Table 2. Excision of dGua-B[a]P adducts from the DNA in BHK At0hr*
At24hr*
Sephadex-LH20 chromatogram
3H/ 4CtoW
% excision
Part At Peak I§ Peak II§
0.529 0.071 0.114
0 0 0
3H/14Ct 0.509 0.044 0.071
At72hr*
% excisiont
3H/14Ct0i
% excisiont
3.8 38.0 37.7
0.630 0.013 0.024
-19.1 81.7 78.9
* Duration of post-treatment incubation. t Reproducibility of data from different experiments is estimated at ±5%.
Sephadex LH-20 part A: fractions 8-20 of 30% methanol/H20 eluate (fraction size: 1.4 ml). methanol/0.035 M Na borate to 80% aqueous methanol/0.01 M Na borate, pH 8.7, gradient containing dGua-B[a]P I. Peak II: fractions 61-73 of the same gradient containing dGua-B[a]P II.
§ Sephadex LH-20 peak I: fractions 50-60 of 30% aqueous
RESULTS Formation and excision of deoxyguanosine-benzo[a]pyrene adducts in BHK An experimental protocol was developed for the determination of the formation and excision of dGua-B[a]P in BHK which allowed the measurement of cell loss during post-treatment incubation, corrections for the yield in the DNA extraction step, assessment of the completeness of the enzymatic hydrolysis of the DNA and of the recovery from the chromatography columns, and corrections for the damage dilution occurring due to de novo DNA synthesis during post-treatment incubation. BHK monolayer cultures were started at low cell densities and were uniformly labeled with ['4C]thymidine in their DNA over five generations. One generation time (i.e., 12 hr) after the initial addition of ['4C]thymidine, [3H]B[a]P was added to a final level of 0.38 ,ug/ml, which had been shown to be essentially nontoxic. Fig. 1 upper shows a typical Sephadex LH-20 chromatogram of a deoxynucleoside hydrolysate obtained from BHK, One major 14C peak and two 3H peaks are discernible in part A of the chromatogram which contains the polar components of the hydrolysate. The '4C peak corresponds to thymidine, the 3H-containing material has not been unambiguously identified. Further analysis by paper electrophoresis only revealed material with the mobility of thymidine, deoxyadenosine, and deoxyguanosine. Two 3H peaks, but no _4C-containing material, were eluted in part B of the Sephadex LH-20 chromatogram. All evidence indicates that peak I mostly corresponds to dGuaB[a ]P I and peak II to the diastereomeric adduct, dGua-B[a ]P II (13-19). These structural assignments are further corroborated by work from our laboratory in which the diastereomeric B[a]P diol-epoxides I and II were synthesized in radioactive form and reacted in vitro with bacteriophage T7-DNA (20). However, chromatographic peaks I and II also may contain minor components such as addition products to deoxyadenosine and deoxycytidine (19, 29). The relative amounts of peaks I and II and the amount of 3H in part A of the chromatogram varied in different experiments and may depend on the exact growth state of the cultures during the exposure to [3H]B[a ]P. In contrast to the work of King et al. (10), in part B of the Sephadex LH-20 chromatogram, only traces of 3H were eluted before peaks I and II regardless of whether our column conditions or those of King et al. (10) were used. The level of total dGuaB[a ]P contained in the DNA of BHK in a typical experiment was estimated at 1.1 residues in 106 deoxyribonucleosides. The fate of the dGua-B[a]P adducts in BHK was followed over 72 hr of post-treatment incubation. For this purpose, the cells~were replated in fresh medium after the removal of [3H]-B[a]P and [14C]thymidine and several washes. The plating
efficiency was approximately 70%, and only 10-15% of the cells were lost from the monolayers during post-treatment incubation as judged by the recovery of I4C label. Part B of representative Sephadex LH-20 chromatograms for 0 and 72 hr of posttreatment incubation are shown in Fig. 2 upper.* It is evident that the 3H/14C total ratio decreased substantially in both peak I and peak II in 72 hr of post-treatment incubation. The data derived from these and analogous chromatograms are shown in Table 2 which contains values for the extent of the removal of dGua-Bfa]P as a function of post-treatment incubation. It follows that approximately 80% of dGua-B[a]P had been removed from the DNA during the 72-hr incubation and that the two stereoisomeric adducts disappeared with identical kinetics. In contrast, the ratio of total 3H contained in part A of the chromatogram to total '4C remained constant during posttreatment incubation.
Formation and excision of deoxyguanosine-benzo[a]pyrene adducts in secondary MEF In contrast to BHK, addition of [3H]B[a]P at 0.33 ,g/ml caused 10-20% growth inhibition relative to control cultures as judged by the time required for the cultures to reach confluency. A representative Sephadex LH-20 chromatogram is shown in Fig. 1 lower. Peaks I and II are discernible in part B of the chromatogram, but their relative heights are reversed compared to BHK and only traces of 3H radioactivity were eluted in part A. The extent of deoxyguanosine arylalkylation in typical experiments with MEF was approximately 1.2 dGua-B[a]P residues
for 105 unmodified deoxyribonucleosides-i.e., approximately 10 times higher than for BHK. The protocol for the dGua-B[a ]P excision studies was analogous to that for BHK outlined above. The plating efficiency was nearly 100% and no loss of '4C radioactivity was observed during post-treatment incubation. As for BHK, the ratio of 3H in the fractions of peaks I and II to total '4C decreased, indicating the removal of dGua-B[a]P from DNA. A plot of the experimental data for 0 and 72 hr of post-treatment incubation is presented in Fig. 2 lower. The data derived from these and analogous chromatograms are given in Table 3. It is evident that removal of the adducts occurred but at a slower rate than for BHK. Removal of peak I was significantly slower than removal of peak II. Because of the low amounts of 3H radioactivity in part A of the chromatograms, no reliable information concerning the fate of this material during post-treatment incubation could be obtained for MEF. *
The ratios of the 3H in the individual fractions over the total 14C contained in all fractions of the chromatogram have been plotted. (Note: all 14C radioactivity is contained in part A of the chromatogram.)
982
Biochemistry: Shinohara and Cerutti
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 3. Excision of dGua-Bla IP adducts from the DNA in secondary MEF At 24 hr*
At 0 hr*
At 48 hr*
At 72 hr*
Sephadex-LH20 chromatogram
3H/'4Ct a
% excision
3H/14Ct0a
excisiont
3H/'4Ct0s
excisiont
3H/14Ct0i
% excisiont
Peak It Peak IIt
1.941 0.774
0 0
1.654 0.671
14.8 13.3
1.319 0.466
32.0 39.8
1.302 0.418
32.9 46.0
%
%
* Duration of post-treatment incubation. t Reproducibility of data from different experiments is estimated at +5%.
Sephadex LH-20 peak I: fractions 53-63 of 30% aqueous methanol/0.035 M Na borate to 80% aqueous methanol/0.01 M Na borate, pH 8.7, gradient containing dGua-B[a]P I. Peak II: fractions 64-74 of same gradient containing dGua-B[aJP II.
DISCUSSION Our results demonstrate that both BHK and secondary MEF possess the capability to remove dGua-B[a]P adducts from their DNA during post-treatment incubation. Adduct removal is slow, particularly in MEF. From values from the literature for the DNA contents per cell for these two cell lines, it is estimated that a BHK cell on average removed 1.1 X 104 molecules of dGua-B[a]P and MEF, 4.3 X 104 molecules in 72 hr. The higher level of dGua-B[a ]P in MEF relative to BHK may lead to saturation of the excision system and may explain why product removal was less complete in MEF. The differences in the generation times between the two cell lines may also be a factor responsible for this difference. It is likely that dGua-B[a ]P adducts are removed from the DNA in our experiments during the prolonged treatment of the cultures with [3H]B[a ]P. The possibility that products other than dGua-B[a]P are formed and rapidly removed during the incubation with the drug cannot be excluded. Evidence for the formation of thymine oxidation products in B[a]P-treated BHK has recently been obtained in our laboratory. Thymine damage is induced by indirect action by metabolites of B[a]P and does not involve the covalent attachment of a B[a]P moiety to the heterocyclic nucleic acid base (M. Ide and P. Cerutti, unpublished results). It should be stressed that our experiments do not allow any conclusions concerning the molecular mechanism of the removal from DNA of dGua-B[a]P-e.g., whether these lesions are recognized by a glycosidase or by a specific endonuclease. Only a negligible loss of cells and a minor effect on growth was observed during the 72-hr post-treatment incubation of both cell lines despite the fact that a considerable fraction of dGua-B[a]P remained in DNA during this period. The relative persistence of these lesions at low toxicity may in part explain the extraordinary mutagenicity of the B[a ]P-diol-epoxides in V79 hamster cells (21-25). It is interesting to note in this context that in MEF the removal of dGua-B[a]P I (i.e., the adduct formed by B[a]P-diol-epoxide I) is significantly slower than removal of the stereoisomeric adduct, dGua-B[a ]P II, formed by B[a ]P-diol-epoxide II. The B[a ]P-diol-epoxide I was found to be more mutagenic in V79 cells than the B[a]P-diol-epoxide II by several investigators (21, 25). In conttast to BHK and MEF, in which the formation of both stereoisomeric deoxyguanosine adducts was observed, in vitro reaction of 7,8-dihydroxy7,8-dihydrobenzo[a ]pyrene with DNA in the presence of rat liver microsomes leads only to the formation of dGua-B[a ]P I (10). Weinstein et al. (29) reported that only dGua-B[a]P I was detectable in the RNA of bovine bronchial explants that had been exposed to [3H]B[a ]P. In rodent cells, the removal of other arylalkylation lesions that involve substitution at the N2-exocyclic amino group of deoxyguanosine has been investigated. Such lesions are formed as major lesions by the carcinogens 7-bromomethylbenz[a ]-
anthracene and as minor lesions by activated derivatives of 2-acetylaminofluorene (30, 31). Similar to our results with dGua-B[a]P, it was found that these lesions were removed only slowly or not at all. Model building suggests that the bulky hydrocarbon substituents of these adducts may be accommodated in the minor groove of the double-stranded DNA helix without causing major conformational distortion. Therefore, these lesions may not be recognized efficiently by the cellular repair machinery. In contrast to these findings, relatively rapid removal of radioactivity from the DNA of human lymphoblastoid Raji cells and concanavalin A-stimulated human peripheral lymphocytes that had been treated with radioactive N-acetoxy-2-acetylaminofluorene has been reported by Scudiero et al. (32). Loss of DNA-bound radioactivity in these experiments is mostly due to the removal of the major lesionsnamely, 8-fN-(2-acetylaminofluorenyl)ldeoxyguanosine. Similarly, the adenine lesions N6-(7-methylbenz[a]anthracenyl)-deoxyadenosine formed by 7-bromomethylbenz[a]anthracene were removed more efficiently from the DNA of nondividing peripheral human lymphocytes than were the lesions N2-(7-methylbenz[a ]anthracenyl)deoxyguanosine (31). We are grateful to Mrs. Wendy Dusek for excellent technical assistance. We thank Dr. P. Brookes for valuable advice in the initial phase of this work. This work was supported by U.S. Public Health Service Grant GM 18617 and Contract AT-(40-1)-4155 of the U.S. Energy
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Excision repair of benzo[a]pyrene-deoxyguanosine adducts in baby hamster kidney 21/C13 cells and in secondary mouse embryo fibroblasts C57BL/6J (benzo[aJpyrene-DNA adducts)
KUNIO SHINOHARA AND PETER A. CERUTTI Department of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida, Gainesville, Fla. 32610
Communicated by Bernhard Witkop, January 3, 1977
The formation and excision of benzo[alpyrene-deoxyguanosine adducts in metabolizing baby hamster kidney cells (21/C13) and secondary mouse embryo fibroblasts (C57BL/6J) was investigated. Both diastereomeric adducts, N2--(10-$7ft,8a,9a-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[aJpyrenejyl)deoxyguanosine and N2(10-f7ft,8a,9#-trihydroxy7,8,9,10-tetrahydrobenzo[alpyrenejyl)deoxyguanosine, were detected in both cell lines and both cell lines were capable of excising these lesions, albeit with low efficiency. There is much support for the notion that damage to the chromosomal DNA is a necessary initial step in physical and chemical carcinogenesis. Many carcinogens have been shown unambiguously to be DNA damaging agents (1-3). Most cells have evolved elaborate mechanisms for the processing of DNA damage. In general, prereplication excision repair-i.e., repair completed before the replication fork reaches the damaged residues8appears to accomplish complete restitution of the structure and activities of the DNA in prokaryotic and eukaryotic cells (4). Biological effects of DNA damaging agents such as cell death, mutation, and malignant transformation appear to be consequences of DNA replication on templates containing unexcised lesions. Therefore, the capacity of a cell to remove damaged residues from DNA by prereplication repair is expected to affect the magnitude of the biological effects (5, 6). The structural features of unexcised lesions may in turn affect the lethal, mutagenic, and carcinogenic efficiencies of the damaging agent (e.g., see refs. 7 and 8). Benzo[a]pyrene (B[a]P) is an ubiquitous pollutant of our environment and is suspected to be carcinogenic in man (9). It has been shown that covalent adducts are formed between B[a]P and DNA in metabolizing mammalian cells, and there is strong evidence that the major products formed are the stereoisomeric N2-(10-1713,8a,9a- or 9f-trihydroxy-7,8,9,10tetrahydrobenzo[a Jpyrenelyl)deoxyguanosines (dGua-B[a ]P I or dGua-B[a]P II, respectively) (10-14). The active B[a]P metabolites reacting with the exocyclic amino group of deoxyguanosine during formation of these adducts are the diastereomeric (+)7f.,8a-dihydroxy-9a,10a or 9,B,10#-epoxy7,8,9,10-tetrahydrobenzo[a]pyrenes (13-20) (B[a]P-diol-expoxides I or II, respectively). It has recently been shown that the B[a ]P-diol-epoxides are exceptionally potent mutagens in Salmonella and in V79 hamster cells (21-25). According to the concepts discussed above, it is expected that a determining Abbreviations: B[alP, benzo[a]pyrene; dGua-B[a]P I, N2-(10J7f,8a,9a-trihydroxy-7, 8,9, 10-tetrahydrobenzo[a pyrenelyl)deoxyguanosine; dGua-B[a]P II, N2-(10-t7B,8a,9fl-trihydroxy-7,8,9,1O-tetrahydrobenzo[a pyrenejyl)deoxyguanosine; B[a]P-diol-epoxide I, (+)7fB,8a-dihydroxy-9a,10a-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-diol-epoxide II, (b)7j6,8a-dihydroxy-9fl,10B-epoxy7,8,9,10-tetrahydrobenzo[ajpyrene; BHK, baby hamster kidney cells; ABSTRACT
MEF, mouse embryo fibroblasts.
979
factor for the biological effects of B[a]P, besides quality and quantity of metabolic activation, is the capacity of a cell to remove the B[a ]P-induced DNA lesions by prereplication excision repair. We are reporting our studies of the removal of dGuaB[a ]P adducts, during post-treatment incubation, from the DNA of baby hamster kidney cells (21/C13) (BHK) and secondary mouse embryo cells (C57BL/6J) (MEF). It was found that both cell lines were able to remove these lesions from their DNA, albeit slowly. The relative persistence of dGua-B[a]P lesions may be a factor in the mutagenic activity of the B[a] P-diol-epoxides. MATERIALS AND METHODS Chemicals. Generally labeled [3H]benzo[a]pyrene (20 Ci/ mmol) was purchased from Amersham/Searle, Arlington Heights, Ill., and dissolved in dimethyl sulfoxide at a concentration of 25 mCi/3.2 ml. [2-14C]Thymidine (61 mCi/mmol) was supplied by the same company. Cell Cultures. BHK were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 10% tryptose phosphate broth solution (29.5 g/liter) under 10% CO2 at 100% humidity. Primary monolayer cultures of MEF were prepared from 16- to 18-day-old C57BL/6J mouse embryos; 5 X 106 cells were inoculated in T150 Corning plastic culture flasks and grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum under 5% CO2 at 100% humidity. Three days later, the cells were harvested and replated at 1 X 106 cells per T150 flask for the experiments. Benzo[ajpyrene Treatment and Post-Treatment Incubation. Approximately 3 X 105 BHK were inoculated into T150 culture flasks, 30 ml of medium was added, and the cultures were incubated for 30-36 hr. Under our growth conditions, BHK have an approximate generation time of 12 hr. Radioactive thymidine (6 nCi of [2-14C]thymidine), cold thymidine (0.03 ,tmol), and cold deoxycytidine (0.03 Mumol) were then added to each culture flask and the same additions were repeated four times at 12-hr intervals. Together with the second addition of [14C]thymidine, 0.1 ml of [3H]B[a]P stock solution in dimethyl sulfoxide was added (final concentration, 0.33 ,ug of B[a ]P per ml growth medium). The cells were washed with fresh prewarmed medium containing 1 ,uM cold thymidine 48 hr after the addition of [3H]B[a]P. The protocol for labeling of BHK with [2-14C]thymidine and treatment with [3H]B[a]P is further described in Table 1. Under these conditions the cultures were almost confluent, the cellular DNA was uniformly labeled with [14C]thymidine, and B[a]P was almost completely metabolized to water-soluble derivatives. The cells were harvested by trypsinization, washed twice, and replated into T150 flasks at I to 2 X 106 cells per flask, and growth was continued up to 72 hr in medium containing 1 MM cold thymidine. After
Biochemistry: Shinohara and Cerutti
980
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 1. Protocol for labeling of BHK with [2- lC ] thymidine and treatment with [3H ]benzo[a ]pyrene Time, hr
Step
0 36 48
5 x 10 BHK in 30 ml of growth medium Add: [4C ]dThd; cold dThd; cold dCyd Add: [4C ]dThd; cold dThd; cold dCyd;
Same as 36 hr Same as 36 hr Same as 36 hr Cells washed, harvested, and replated for post-treatment incubation
post-treatment incubation, the cells were harvested and washed several times with Hanks' balanced salt solution. Analogous procedures were used for MEF except that the labeling protocol was adjusted to the generation time of approximately 24 hr. At the outset of the experiment, the cells were in their second passage. [2-'4C]thymidine (20 nCi), cold thymidine (0.03 ,umol), and cold deoxycytidine (0.03,umol) were added three times in 24-hr intervals. Radioactive B[a 1P (final concentration, 0.33 tg/ml of growth medium) was added with the second addition of ['4C]thymidine. The B[a]P was almost completely metabolized to water-soluble derivatives within 48 hr. The cells were harvested, washed, replated, and subjected to -post-treatment incubation as described for BHK. DNA Extraction and Enzymatic Hydrolysis. The methods for DNA extraction were those of Diamond et al. (26) and Duncan et al. (27), and the conditions for the digestion of the DNA to nucleosides were those described by Baird and Brookes B
1500 - A
BHK -
I
2500 -2500 n
5o
I
ON
[3HJB[a]P
60 72 84 96
E 1000
BHK
u
I ___ I_
a II p . 20 0
3
h-
-
20
40
60
80
0
300
I.
E
-50 3
I FRACTION NUMBER
FIG. 1. Sephadex LH-20 chromatograms of DNA--digests from BHK (Upper) and secondary MEF (Lower) that had be-en exposedto see 1[4C]thymidine and [3H]B[a]P (for experimental prn Materials and Methods). Peaks I and II in part B of ea ch chromatogram correspond to the deoxyguanosine-benzo[a]pyr -ene adducts, dGua-B[a]P I and dGua-B[a]P II. The horizontal bar, sgive the position of elution of 4-p-nitrobenzylpyridine which was added as absorbancy marker to each sample.
,cedures,
0.
o20 10,
I
B
40
60 ~~~~~~I
80
MEF
0.4 0.3-
0.2-
0.
20 60 40 FRACTION NUMBER
80
FIG. 2. Removal of dGua-BlaJP I and dGua-B[a]P II from the DNA of BHK and secondary MEF during post-treatment incubation. Part B of Sephadex LH-20 chromatograms of DNA digests from BHK (Upper) and secondary MEF (Lower) that had been exposed to [14C]thymidine and [3H]B[a]P and then incubated in fresh medium for 0 and 72 hr. The ratios of the 3H radioactivity contained in each fraction to total 14C radioactivity eluted from the column are plotted (note that all 14C radioactivity is contained in part A of the chromatogram as is shown in Fig. 1). The horizontal bars give the position of elution of 4-p-nitrobenzylpyridine which was added as absorbancy marker to each sample.
(28). The following minor modifications were made: (i) the extracted DNA was precipitated once with ethanol; (ii) double the amounts of hydrolytic enzymes were used for DNA digestion; and (iii) phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) was added in two installments at the beginning and after 24 hr of the time allowed for digestion. Under these conditions, the 14C label was renderedcompletely soluble in 10% (wt/vol) trichloroacetic acid. The 3H label was not completely solubilized. This is attributed to the limited solubility of benzo[a]pyrene-substituted nucleosides in trichloroacetic acid. No evidence for the formation of oligonucleotide limit digests was obtained from the Sephadex LH-20 chromatograms (see below). Sephadex LH-20 Chromatography. A small amount of 4p-nitrobenzylpyridine was added to the DNA digests as 'absorbance marker and the samples were applied to 0.9 X 24 cm Sephadex LH-20 columns. The columns were first eluted with 30% (vol/vol) aqueous methanol and twenty-four 1.4-ml fractions were collected. The columns were further eluted with a linear gradient of 30% aqueous methanol/sodium borate to 80% aqueous methanol/sodium borate. The starting solvent was prepared by adding 26 ml of methanol to 61 ml of 0.05 M sodium borate at pH 8.7, and the final solvent contained 80 ml of methanol and 20 ml of 0.05 M sodium borate at pH 8.7. Seventy-six 1.4-ml fractions were collected and their radioactivity was counted in a Beckman model LS 233 scintillation system after the addition of 14 ml of Aquasol to each fraction.
Recovery of 14C radioactivity from the columns was always
complete; the recovery of 3H radioactivity was 90-100%.
Biochemistry: Shinohara, and Cerutti
Proc. Natl. Acad. Sci. USA 74 (1977)
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Table 2. Excision of dGua-B[a]P adducts from the DNA in BHK At0hr*
At24hr*
Sephadex-LH20 chromatogram
3H/ 4CtoW
% excision
Part At Peak I§ Peak II§
0.529 0.071 0.114
0 0 0
3H/14Ct 0.509 0.044 0.071
At72hr*
% excisiont
3H/14Ct0i
% excisiont
3.8 38.0 37.7
0.630 0.013 0.024
-19.1 81.7 78.9
* Duration of post-treatment incubation. t Reproducibility of data from different experiments is estimated at ±5%.
Sephadex LH-20 part A: fractions 8-20 of 30% methanol/H20 eluate (fraction size: 1.4 ml). methanol/0.035 M Na borate to 80% aqueous methanol/0.01 M Na borate, pH 8.7, gradient containing dGua-B[a]P I. Peak II: fractions 61-73 of the same gradient containing dGua-B[a]P II.
§ Sephadex LH-20 peak I: fractions 50-60 of 30% aqueous
RESULTS Formation and excision of deoxyguanosine-benzo[a]pyrene adducts in BHK An experimental protocol was developed for the determination of the formation and excision of dGua-B[a]P in BHK which allowed the measurement of cell loss during post-treatment incubation, corrections for the yield in the DNA extraction step, assessment of the completeness of the enzymatic hydrolysis of the DNA and of the recovery from the chromatography columns, and corrections for the damage dilution occurring due to de novo DNA synthesis during post-treatment incubation. BHK monolayer cultures were started at low cell densities and were uniformly labeled with ['4C]thymidine in their DNA over five generations. One generation time (i.e., 12 hr) after the initial addition of ['4C]thymidine, [3H]B[a]P was added to a final level of 0.38 ,ug/ml, which had been shown to be essentially nontoxic. Fig. 1 upper shows a typical Sephadex LH-20 chromatogram of a deoxynucleoside hydrolysate obtained from BHK, One major 14C peak and two 3H peaks are discernible in part A of the chromatogram which contains the polar components of the hydrolysate. The '4C peak corresponds to thymidine, the 3H-containing material has not been unambiguously identified. Further analysis by paper electrophoresis only revealed material with the mobility of thymidine, deoxyadenosine, and deoxyguanosine. Two 3H peaks, but no _4C-containing material, were eluted in part B of the Sephadex LH-20 chromatogram. All evidence indicates that peak I mostly corresponds to dGuaB[a ]P I and peak II to the diastereomeric adduct, dGua-B[a ]P II (13-19). These structural assignments are further corroborated by work from our laboratory in which the diastereomeric B[a]P diol-epoxides I and II were synthesized in radioactive form and reacted in vitro with bacteriophage T7-DNA (20). However, chromatographic peaks I and II also may contain minor components such as addition products to deoxyadenosine and deoxycytidine (19, 29). The relative amounts of peaks I and II and the amount of 3H in part A of the chromatogram varied in different experiments and may depend on the exact growth state of the cultures during the exposure to [3H]B[a ]P. In contrast to the work of King et al. (10), in part B of the Sephadex LH-20 chromatogram, only traces of 3H were eluted before peaks I and II regardless of whether our column conditions or those of King et al. (10) were used. The level of total dGuaB[a ]P contained in the DNA of BHK in a typical experiment was estimated at 1.1 residues in 106 deoxyribonucleosides. The fate of the dGua-B[a]P adducts in BHK was followed over 72 hr of post-treatment incubation. For this purpose, the cells~were replated in fresh medium after the removal of [3H]-B[a]P and [14C]thymidine and several washes. The plating
efficiency was approximately 70%, and only 10-15% of the cells were lost from the monolayers during post-treatment incubation as judged by the recovery of I4C label. Part B of representative Sephadex LH-20 chromatograms for 0 and 72 hr of posttreatment incubation are shown in Fig. 2 upper.* It is evident that the 3H/14C total ratio decreased substantially in both peak I and peak II in 72 hr of post-treatment incubation. The data derived from these and analogous chromatograms are shown in Table 2 which contains values for the extent of the removal of dGua-Bfa]P as a function of post-treatment incubation. It follows that approximately 80% of dGua-B[a]P had been removed from the DNA during the 72-hr incubation and that the two stereoisomeric adducts disappeared with identical kinetics. In contrast, the ratio of total 3H contained in part A of the chromatogram to total '4C remained constant during posttreatment incubation.
Formation and excision of deoxyguanosine-benzo[a]pyrene adducts in secondary MEF In contrast to BHK, addition of [3H]B[a]P at 0.33 ,g/ml caused 10-20% growth inhibition relative to control cultures as judged by the time required for the cultures to reach confluency. A representative Sephadex LH-20 chromatogram is shown in Fig. 1 lower. Peaks I and II are discernible in part B of the chromatogram, but their relative heights are reversed compared to BHK and only traces of 3H radioactivity were eluted in part A. The extent of deoxyguanosine arylalkylation in typical experiments with MEF was approximately 1.2 dGua-B[a]P residues
for 105 unmodified deoxyribonucleosides-i.e., approximately 10 times higher than for BHK. The protocol for the dGua-B[a ]P excision studies was analogous to that for BHK outlined above. The plating efficiency was nearly 100% and no loss of '4C radioactivity was observed during post-treatment incubation. As for BHK, the ratio of 3H in the fractions of peaks I and II to total '4C decreased, indicating the removal of dGua-B[a]P from DNA. A plot of the experimental data for 0 and 72 hr of post-treatment incubation is presented in Fig. 2 lower. The data derived from these and analogous chromatograms are given in Table 3. It is evident that removal of the adducts occurred but at a slower rate than for BHK. Removal of peak I was significantly slower than removal of peak II. Because of the low amounts of 3H radioactivity in part A of the chromatograms, no reliable information concerning the fate of this material during post-treatment incubation could be obtained for MEF. *
The ratios of the 3H in the individual fractions over the total 14C contained in all fractions of the chromatogram have been plotted. (Note: all 14C radioactivity is contained in part A of the chromatogram.)
982
Biochemistry: Shinohara and Cerutti
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 3. Excision of dGua-Bla IP adducts from the DNA in secondary MEF At 24 hr*
At 0 hr*
At 48 hr*
At 72 hr*
Sephadex-LH20 chromatogram
3H/'4Ct a
% excision
3H/14Ct0a
excisiont
3H/'4Ct0s
excisiont
3H/14Ct0i
% excisiont
Peak It Peak IIt
1.941 0.774
0 0
1.654 0.671
14.8 13.3
1.319 0.466
32.0 39.8
1.302 0.418
32.9 46.0
%
%
* Duration of post-treatment incubation. t Reproducibility of data from different experiments is estimated at +5%.
Sephadex LH-20 peak I: fractions 53-63 of 30% aqueous methanol/0.035 M Na borate to 80% aqueous methanol/0.01 M Na borate, pH 8.7, gradient containing dGua-B[a]P I. Peak II: fractions 64-74 of same gradient containing dGua-B[aJP II.
DISCUSSION Our results demonstrate that both BHK and secondary MEF possess the capability to remove dGua-B[a]P adducts from their DNA during post-treatment incubation. Adduct removal is slow, particularly in MEF. From values from the literature for the DNA contents per cell for these two cell lines, it is estimated that a BHK cell on average removed 1.1 X 104 molecules of dGua-B[a]P and MEF, 4.3 X 104 molecules in 72 hr. The higher level of dGua-B[a ]P in MEF relative to BHK may lead to saturation of the excision system and may explain why product removal was less complete in MEF. The differences in the generation times between the two cell lines may also be a factor responsible for this difference. It is likely that dGua-B[a ]P adducts are removed from the DNA in our experiments during the prolonged treatment of the cultures with [3H]B[a ]P. The possibility that products other than dGua-B[a]P are formed and rapidly removed during the incubation with the drug cannot be excluded. Evidence for the formation of thymine oxidation products in B[a]P-treated BHK has recently been obtained in our laboratory. Thymine damage is induced by indirect action by metabolites of B[a]P and does not involve the covalent attachment of a B[a]P moiety to the heterocyclic nucleic acid base (M. Ide and P. Cerutti, unpublished results). It should be stressed that our experiments do not allow any conclusions concerning the molecular mechanism of the removal from DNA of dGua-B[a]P-e.g., whether these lesions are recognized by a glycosidase or by a specific endonuclease. Only a negligible loss of cells and a minor effect on growth was observed during the 72-hr post-treatment incubation of both cell lines despite the fact that a considerable fraction of dGua-B[a]P remained in DNA during this period. The relative persistence of these lesions at low toxicity may in part explain the extraordinary mutagenicity of the B[a ]P-diol-epoxides in V79 hamster cells (21-25). It is interesting to note in this context that in MEF the removal of dGua-B[a]P I (i.e., the adduct formed by B[a]P-diol-epoxide I) is significantly slower than removal of the stereoisomeric adduct, dGua-B[a ]P II, formed by B[a ]P-diol-epoxide II. The B[a ]P-diol-epoxide I was found to be more mutagenic in V79 cells than the B[a]P-diol-epoxide II by several investigators (21, 25). In conttast to BHK and MEF, in which the formation of both stereoisomeric deoxyguanosine adducts was observed, in vitro reaction of 7,8-dihydroxy7,8-dihydrobenzo[a ]pyrene with DNA in the presence of rat liver microsomes leads only to the formation of dGua-B[a ]P I (10). Weinstein et al. (29) reported that only dGua-B[a]P I was detectable in the RNA of bovine bronchial explants that had been exposed to [3H]B[a ]P. In rodent cells, the removal of other arylalkylation lesions that involve substitution at the N2-exocyclic amino group of deoxyguanosine has been investigated. Such lesions are formed as major lesions by the carcinogens 7-bromomethylbenz[a ]-
anthracene and as minor lesions by activated derivatives of 2-acetylaminofluorene (30, 31). Similar to our results with dGua-B[a]P, it was found that these lesions were removed only slowly or not at all. Model building suggests that the bulky hydrocarbon substituents of these adducts may be accommodated in the minor groove of the double-stranded DNA helix without causing major conformational distortion. Therefore, these lesions may not be recognized efficiently by the cellular repair machinery. In contrast to these findings, relatively rapid removal of radioactivity from the DNA of human lymphoblastoid Raji cells and concanavalin A-stimulated human peripheral lymphocytes that had been treated with radioactive N-acetoxy-2-acetylaminofluorene has been reported by Scudiero et al. (32). Loss of DNA-bound radioactivity in these experiments is mostly due to the removal of the major lesionsnamely, 8-fN-(2-acetylaminofluorenyl)ldeoxyguanosine. Similarly, the adenine lesions N6-(7-methylbenz[a]anthracenyl)-deoxyadenosine formed by 7-bromomethylbenz[a]anthracene were removed more efficiently from the DNA of nondividing peripheral human lymphocytes than were the lesions N2-(7-methylbenz[a ]anthracenyl)deoxyguanosine (31). We are grateful to Mrs. Wendy Dusek for excellent technical assistance. We thank Dr. P. Brookes for valuable advice in the initial phase of this work. This work was supported by U.S. Public Health Service Grant GM 18617 and Contract AT-(40-1)-4155 of the U.S. Energy
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