[CANCER RESEARCH 42, 3480-3485, September 1982] 0008-5472/82/0042-0000$02.00 Role of Depurination in Mutagenesis by Chemical Carcinogens1
Roeland M. Schaaper,2 Barry W. Glickman, and Lawrence A. Loeb3
The Joseph Gottstein Memoria/ Cancer Research Laboratory, Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 98195 {R. M. S., L. A. L.], and Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [B. W.G.]
ABSTRACT significant extent during the presumably modified mode of replication in SOS-induced bacteria. In these experiments, The effect of modifying
INTRODUCTION Chemicals. BPL was obtained from Sigma Chemical Co. (St. Louis, Mo.); [3H]aBDPE (565 mCi/mmol) and [3H]NAAAF (710 mCi/mmol) To understand the process of chemical carcinogenesis at were obtained from Midwest Research Institute, Kansas City, Mo., the molecular level, a detailed knowledge is required of the through the Cancer Research Program of the Division of Cancer Cause interaction of chemical carcinogens with cellular macromole- and Prevention, National Cancer Institute, Bethesda, Md. Bacteria and Bacteriophage. <;>x174single-stranded viral DNA and cules, in particular with DNA. This interaction, frequently in the RF I DNA were prepared as described previously (8). High-titer stocks form of covalent binding to the DNA, is thought to be a crucial of .-.Xam3 and am86 were prepared as described (8). ' am3 is located initiating event in carcinogenesis (14). Progress in this field has at positions 586 to 588 in gene E(D), and am86 is located at positions been hampered by the complex variety of lesions produced in 4116 to 4118 in gene A (17). E. coli HF4714 (si/-7+) and HF4704 DNA by most of these agents. It has therefore been difficult to (su") were used as indicator strains for determining phage reversion assign the mutagenic or carcinogenic effects of an agent to frequencies. one particular lesion. We have recently developed a combined Transfection Procedures. Spheroplasts were prepared from E. coli in vitro-in vivo assay using x174 DNA, which allows the DNA KT-1 through the lysozyme-EDTA procedure (8, 20). For SOS induc to be treated in vitro and to be introduced into the cell by tion, the bacteria were irradiated in thin layers with UV (80 J/sq m) (254 nm) and incubated for 45 min at 37°prior to their conversion to transfection. This permits us to control both the degree of damage to the DNA and the condition of the host. This tech spheroplasts (20). The details of the transfection procedure have been described (8). In short, 1.Oftg
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-70° and thawed and 100 id chloroform had been added, phage titer and reversion frequency of the resultant progeny phage were deter mined by plating on HF4714 (su+) and HF4704 (su"). Modification of the DNA. One volume of o,\l 74 ONA (0.1 /ig/ ml in 10 ÕTIMTris-HCI, pH 8.0; 0.1 mM EDTA) was modified by adding 0.1 volume of an appropriate dilution of the activated carcinogen. Incuba tion was at 37°for 1 to 2 hr in the dark to prevent photodecomposition. This DNA was either used directly or stored at -70°. BPL was diluted in 20 mM Tris (pH 8.0) immediately before use. aBPDE was diluted in dimethylformamide, and NAAAF was diluted in dimethyl sulfoxide. Depurination of unmodified <|>x174 DNA was performed at pH 5.00 and 70°as described (20). Heat treatment subsequent to modification was by addition of 10 volumes of 10 mM potassium phosphate buffer (pH
7.0) followed by incubation at 70°for 1 hr in the dark. 0200 KXDO 2000 5 Characterizing Heat-induced Sites. Alkali-labile sites were demon Cone(juMI Chart 1. Inactivation of the plaque-forming capacity of <(>x174single-stranded strated by treating the modified and/or heated DNA with an equal DNA upon treatment with increasing concentrations of BPL, NAAAF, and aBPDE. volume of 0.2 N NaOH at 37°for 20 min followed by sucrose gradient Curves are averages of 4 to 10 different experiments. See "Materials and analysis. Neutral sucrose gradients (5 to 20%) were run for 4.5 hr at Methods" for details of the transfection procedure. Cone., concentration. 40,000 rpm in a SW 50.1 rotor. The identity of apurinic sites was established by using the "nicked-circle assay" as developed by Kuhn- lein ef al. (7). The assay measures the conversion of RF I to replicative form II DNA which results in the DNA sticking to nitrocellulose filters upon quick denaturation and renaturation. To measure apurinic sites, they must be hydrolyzed by alkali treatment (30 min, 37°,0.1 N NaOH) or enzymatically by treatment with apurinic endonucleases. The latter was conducted by incubating 0.5 ft 0x1 74 RF I (1 1,000 cpm 3H per /ig) in a final volume of 150 /il, containing 25 mM Tris-HCI (pH 7.50), 0.2 mM EDTA, 12.5 mM MgCI2, and 0.005% Triton X-100, with 1 jtl apurinic endonuclease purified from HeLa cells (20 units/fil) for 30 min at 37°, terminated by the addition of EDTA to 20 mM. The apurinic endonuclease was kindly provided by Drs. C. M. Kane and S. Linn (5).
RESULTS Chart 2. Increase in reversion frequency of
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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. R. M. Schaaper et al. within each experiment, the relative mutagenicities of the 4 treatments are similar. It can be concluded that heat-acid is am 86 generally the most effective in eliciting mutagenesis expressed per lethal hit. This is striking with am3 but is also observed with am86. Effect of Heat on Survival and Mutagenesis. Several differ ent base modifications, most notably at positions A/3and A/7of purines, destabilize the A/-glycosylic bond and increase the rate of depurination (11 ). To assess whether depurination through this pathway could contribute to mutagenesis by the agents used in this study, we heated the modified 0x174 DNA molecules at 70°at neutral pH. This procedure is expected to effectively hydrolyze the labilized bases (11) without causing significant depurination of unmodified bases. For aBPDE- and NAAAF-modified DMAs, no detectable changes in either sur vival or mutation frequencies could be detected [within nor mal confidence levels (changes less than 2-fold; results not IOOO 0 IOO 500 IOOO shown)]. In contrast, in the case of BPL-modified DNA, a strong Cane (juM) inactivation occurred as a result of heat treatment (Charts 3 Chart 4. Reversion frequencies of BPL-treated am3 and am86 DNA before and 4). This inactivation was reproducible in several different and after heat treatment as measured in SOS-induced cells. See legend to Chart 3. The increase for control am3 DNA might be due to direct depurination (about experiments and corresponded to the introduction of 3 addi one-third of an apurinic site is expected per molecule). Cone., concentration. tional lethal hits for every preexisting lethal hit. It is unlikely that this inactivation was due to increased reaction of the DNA with 4000 residual amounts of BPL since the samples were routinely diluted 10-fold before heating, and even overnight dialysis against a large volume of buffer prior to heating did not diminish the heat-induced inactivation. Heating BPL-modified DNA also has a large effect on the reversion frequencies of am3 and 3000 am86 (Chart 4). A 7- to 9-fold increase is observed for the 2 mutants.
2000
normal IOOO
O I 2 3 4 5 ml into gradient Chart 5. Neutral sucrose gradient analysis of BPL-modified .>\ i74 single- stranded DNA. Fractions were collected from the top. Sedimentation is from left to right. Arrow, position of the x174single-stranded circle. The DNA was treated with 320 HIMBPL. The modified DNA does not shift its position (not shown). Indicated are the profiles obtained with this DNA subsequently heated (•)or alkali treated (O), or both (A). Neither heat, nor alkali, nor their combination affected the position of unmodified DNA (not shown).
Analysis of Modified-Heated DNA. Chart 5 shows a neutral sucrose gradient analysis of the BPL-modified DNA before and after exposure to elevated temperature. BPL-modified DNA sediments as an intact single-stranded circle as does the same DNA treated with 0.1 N NaOH. This indicates that few if any breaks and/or alkali-labile sites are present in this DNA. Ex posure of BPL-modified DNA to elevated temperature fails to introduce single-stranded breaks. However, the heat treatment does produce alkali-labile sites. Since these heat-induced al Chart 3. Inactivation of the plaque-forming ability of BPL-modified <)>x174 single-stranded DNA before and after treatment for 1 hi at 70°(10 HIMpotassium kali-sensitive sites could well be apurinic sites, their identity was further investigated in the "nicked-circle" assay for apu- phosphate buffer, pH 7.00). Cone., concentration.
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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Depurination by Chemical Carcinogens rinic sites as developed by Kuhnlein et al. (7). The results are Replicating complex given in Table 2. In this assay, it is necessary to use covalently Bulky closed circular double-stranded DMA. Prior to heat treatment, irrLpl x adduci few apurinic sites are present in the modified DMA (0.12), and i 111111.i iiii 111111tAj '''' heat treatment alone causes few strand breaks (0.22). How DNA Replication Stoppage ever, heat treatment of the modified DMA results in 1.90 apu rinic sites/circle as measured by hydrolysis with an apurinic Spontaneous endonuclease. This is indistinguishable from the number of depurination alkali-sensitive sites in this DMA (1.83). In one other experiment (not shown), the number of apurinic sites was at least 70% of the total alkali-sensitive sites. , Cleavage Poised Altered by DNA synthesis replicating complex glycosylase DISCUSSION Chart 6. Mutagenesis by chemical carcinogens via depurination. In this paper, we report that the in vitro modification-trans- fection system of $x174 DNA can be applied to study muta- such a system (18, 19). We propose a possible mechanism by genesis by chemical carcinogens. All 3 tested carcinogens which apurinic sites function as intermediates in the mutagen were found to be mutagenic in this system. In trying to define esis by many chemical carcinogens, including those that cause the lesions specifically responsible for the mutagenic and/or a (transient) termination of DNA synthesis. Apurinic sites are carcinogenic effects of certain agents, this system may offer a attractive as such intermediates since they are the potential number of advantages. The use of single-stranded DNA allows secondary product of a large variety of lesions. Moreover, one to analyze the results more directly with respect to the when SOS has been induced, they are mutagenic in vivo (20) outcome of the interaction of DNA replication with damaged and are copied to measurable extents by DNA polymerases in bases with little interference by DNA repair. The use of a phage vitro4 In its simplest form, this model (Chart 6) involves cessa system allows one to analyze the response in the absence or tion of DNA replication at bulky adducts and the resultant presence of "inducible error-prone repair." Moreover, in vitro induction of an error-prone (SOS) response (18, 19); and, as DNA modification opens up the possibility of further in vitro a consequence, the replication complex may be modified so manipulations, like heat, acid, or alkali treatments or enzymatic as to more easily replicate past modified DNA structures. In treatments such as by A/-glycosylase or damage-specific en- case of specific adducts, their depurination, either sponta donucleases. To exploit the full potential of this strategy, a neously or enzymatically by specific A/-glycosylases, could basic knowledge of the different kinds of DNA damage and provide a much better substrate for error-prone bypass than their chemical properties is needed. Such knowledge is becom does the original blocking lesion. Thus, apurinic sites may ing increasingly available. A limitation of the system in its represent an important intermediate in the production of mu present form is that it measures only base substitution muta tations. We speculate that the presence of polymerase may tions, ignoring the potentially important contribution of base protect the apurinic site from hydrolysis by endonuclease prior addition-deletion mutations. to DNA replication. Mutagenesis by the 3 chemical carcinogens used in this To explore whether depurination of labilized bases could study proved to be SOS dependent. Therefore, the lesions play a role in mutagenesis by the agents used here, we sub responsible for mutagenesis are not likely to cause direct jected the modified DNA to moderate heat treatment at neutral miscoding but rather to result in termination of normal DNA pH. This treatment had only a minor effect on either survival or synthesis. One might therefore speculate on the importance of mutation frequency of the undamaged DNA or the DNA treated ¡nducibleerror-prone systems (like the E. coli SOS system) in with aBPDE or NAAAF. In contrast, both survival and mutation mutagenesis and/or carcinogenesis by these compounds in frequency of BPL-treated DNA were strongly affected. Two mammalian cells. Studies with SV40 point to the existence of explanations can be advanced for the absence of an effect in the case of aBPDE or NAAAF. (a) Since both the bulky adducts Table 2 produced by these carcinogens and subsequent apurinic sites Number of apurinic sites in BPL-modified DNA: effect of heat treatment The nicked-circle assay is described in "Materials and Methods." 3H-labeled are likely to block DNA synthesis and therefore constitute lethal 0x174 RF I DNA was used, either normal or BPL modified. The total number of lesions, transformation of one into the other would change counts bound to the nitrocellulose filter was 2100. Assay is in single points. A neither survival nor mutagenicity if the lethal or mutagenic second experiment yielded similar results. potentials of the 2 types of lesions were approximately equal, No. of counts bound to nitrocellulose (b) Heating the modified DNA might not depurinate the ad- filter ducted bases very efficiently. aBPDE and NAAAF interact BPLNoneTreatment following BPL589 (1.6mw)400 predominantly at positions N2 and C8 of guanine (3). These (ND)a'fi (ND) modifications do not labilize the glycosylic bond. In the case of Apurinic endonuclease 595 (ND) 740(0.12) aBPDE, modification at position N7 of guanine occurs as a Heat 490 (ND) 860 (0.22) minor product (10 to 20%), and an alkaii-labile adenine product Alkali' 800(0.17) 770(0.15) Heat + alkali' 790(0.15) 1859(1.83) has also been detected (6, 16). It is conceivable that depuri Heat + apurinic endonucleaseNo 580 (ND)BPL 1875(1.90) nation of these minor products has minor effects on mutation * ND, nondetectable (less than 0.05 site). " Numbers in parentheses, calculated numbers of apurinic sites present per frequencies, but the sensitivity of the 0x174 assay is such that changes up to 2-fold cannot be assessed unambiguously. molecule. c Measures alkali-sensitive sites rather than apurinic sites. Significant changes are observed upon heat treatment of
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BPL-modified DNA. Our contention that the increased muta- present in vitro, potentially altering the molar concentration. genesis observed upon heat treatment of BPL-modified DMA is This bacterial "sieving" effect is likely to vary among different likely to result from depurination is based on the following carcinogens. Therefore, comparisons are more appropriate on arguments, (a) Depurination is expected since, of the 4 char the basis of final DNA modification than on a molar basis, (o) acterized BPL DNA adducts [i.e., the (2-carboxyethyl) addition Because only a single Salmonella tester strain was used, the product to the A/7 position of guanine, the A/1 position of quantitative results depend on the mutational specificity of the adenine, and the A/3 positions of both cytosine and thymine], agents tested, (c) DNA modification in vivo elicits DNA repair, the A/7-guanine product is the most frequent (70%) (21), and which is likely to be different for different kinds of damage. its depurination has been well established (1, 21 ). It is important Such effects could readily allow agents producing similar to note, however, that limited depurination of the A/1-adenine amounts of apurinic sites in vitro to show widely different product has also been reported (1). (b) Heat introduces 3 mutagenicities in vivo, (d) Most importantly, mutagenesis at additional lethal hits for every preexisting lethal hit. Apurinic apurinic sites is strongly SOS dependent. Therefore, the ability sites have been shown to be lethal hits (20). (c) BPL-treated of the compound to induce the SOS response may be rate DNA is essentially alkali stable. Upon treatment, however, limiting in mutagenesis. This will depend both on the qualities alkali-sensitive sites are observed. Apurinic sites are alkali of the lesions and on their quantities, which in turn are influ sensitive (13). (d) Filter-binding studies with apurinic endonu- enced by repair processes. clease show that at least 70% of the heat-induced alkali-sen The <>x174 system as used in our study has the potential to sitive sites have the characteristics of apurinic sites. However, circumvent each of these problems and should therefore be this last argument must be qualified since the experiment was well suited for the described methods of analysis. The differ done with double-stranded DNA and the product distributions ential results of aBPDE- and NAAAF- compared to BPL-treated could be different for double- and single-stranded DNA. DNA, however, demonstrate the limitations of any simplistic It may prove instructive to make some calculations based on modeling. Mutagenesis by these compounds probably occurs the survival and mutagenesis data given in Charts 3 and 4. It through a number of different pathways. Depurination could appears that heat treatment of BPL-modified DNA is equally play a minor role in mutagenesis by some agents but a major mutagenic for am3 and am86 DNA since, corrected for back role in mutagenesis by compounds like BPL or aflatoxin Bn ground reversion frequencies, an approximately equal increase where the major product is adduction at the N7 position of is observed (7- and 9-fold, respectively). However, in terms of guanine (25). These studies show the potential available when absolute increases, am86 is much more mutable than is am3. DNA can be treated in vitro, subsequently modified by a variety Calculating the mutagenicities per lethal hit before and after of means, and then used to transfect host cells of a chosen heat treatment, one obtains the following values: am3, 8 x repair capacity. 10"6 (before) and 10 x 10~6 (after); am86, 23 x 10~6 (before) and 55 x 10~6 (after). The analogous data for directly depuri- nated DNA in the same experiment were 32 x 10~6 (am3) and REFERENCES
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Roeland M. Schaaper, Barry W. Glickman and Lawrence A. Loeb
Cancer Res 1982;42:3480-3485.
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