Role of Depurination in Mutagenesis by Chemical Carcinogens1

Home , Depurination

[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 x"174viral DMA by the chemical apurinic sites were introduced by a combined acid-heat treat carcinogens /?-propiolactone, A/-acetoxyacetylaminofluorene ment. Although a role for other heat-acid-induced lesions can and anf/-benzo[a]pyrene diol-epoxide was investigated by not be rigorously excluded, the mutagenicity of apurinic sites transfecting the modified DMA into Escherichia coli sphero- is strongly supported by the abolition of mutagenesis by alkali plasts. Modification of the DMA in vitro by each of these agents (20) and by purified apurinic endonuclease.4 Apurinic sites was mutagenic for the <(>x174amber mutants am3 and am86. could also be important for mutagenesis by chemical carcino Mutagenicity depended on the induction of the "SOS" re gens. Modification of purines at A/3 and A/7 and of pyrimidines sponse in the host spheroplasts. Heating ÃŸ-propiolactone- at O2 positions labilizes the /V-glycosylic bond, leading to treated DNA at neutral pH caused strong inactivation such that sharply increased spontaneous depurination rates (2, 11, 22). the number of lethal hits was increased 4-fold. Sucrose gra Alternatively, apurinic sites may be created in vivo by W-gly- dient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded cosylases, of which increasing numbers are being discovered (12, 23). X174DNA showed greater than 70% of these sites to be In this paper, we used this <>x174 transfection system to apurinic sites. Concomitantly with the production of these new evaluate the lethal and mutagenic consequences of treatment sites, a strong increase in the mutation frequency was ob of 0X-DNA with the chemical carcinogens BPL,5 NAAAF, and served. This mutagenesis also depended upon the induction of aBPDE. We report that each of these agents is mutagenic when the error-prone SOS response in the spheroplasts, as was error-prone repair has been induced. In the case of BPL, we previously shown to be the case for mutagenesis at putative present evidence suggesting that the mutagenic consequences apurinic sites induced directly by acid-heat treatment. These of modification may be enhanced by the creation and bypass results suggest that depurination may be of importance to the of apurinic sites. mechanism of mutagenesis by /3-propiolactone and other car cinogens. MATERIALS AND METHODS

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 37Â°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 x174DNA, either normal or carcinogen- nique has allowed us to establish the mutagenicity of apurinic modified, was dissolved in 5 ml 20 mw Tris-HCI (pH 8.1 ). Spheroplasts sites (20) in Escherichia coli spheroplasts in which the error- (5 ml) were added, and the mixture was incubated at 37Â°for 15 min. prone SOS response had been induced. No mutagenic re At that time, 10 ml prewarmed PAM medium (pH 7.2), containing 10 sponse was observed in normal spheroplasts. These results g/liter nutrient broth, 10 g/liter casamino acids, 10 g/liter glucose, were interpreted to mean that apurinic sites cannot easily be 100 g/liter sucrose, and 0.2% MgSO4 were added, and incubation copied during normal DNA replication but are copied to a continued for approximately 90 min. This entire procedure was per formed under subdued light. After the mixture was frozen at â€”20Â°or ' This investigation was supported by grants from NIH (CA-24845, Ca-24998. and AG-01751) and the National Science Foundation (PCM-76-80439). ' R. M. Schaaper and L. A. Loeb. Infidelity of ONA synthesis associated with 2 Present address: Laboratory of Molecular Genetics, National Institute of bypass of apurinic sites, submitted for publication. Environmental Health Sciences, Research Triangle Park, N. C. 27709. 5 The abbreviations used are: BPL, /i-propiolactone; NAAAF, N-acetoxyace- 3 To whom requests for reprints should be addressed. tylaminofluorene: aBPDE, (Â±)ai7f/-benzo[a]pyrene diol-epoxide; RF I, replicative Received December 31, 1981 ; accepted May 28. 1982. form I.

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-70Â° 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 Ã•TIMTris-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 37Â°for 1 to 2 hr in the dark to prevent photodecomposition. This DNA was either used directly or stored at -70Â°. 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 70Â°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 70Â°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 37Â°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, 37Â°,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 37Â°, 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 x174am3 upon treatment with BPL, aBPDE, and NAAAF or upon depurination by heat-acid treatment. Trans fection was either in normal spheroplasts (â€¢)or in SOS-induced spheroplasts Inactivation and Mutagenesis of <;>x174 DNA. The loss of (O). The higher dose values for BPL in normal spheroplasts were not recorded in biological activity by treatment of single-stranded x174 DNA this particular experiment. Other experiments (not shown) confirmed the absence with increasing concentrations of BPL, aBPDE, and NAAAF is of an appreciable response. Cone., concentration. displayed in Chart 1. These carcinogens interact directly with Table 1 DNA and do not require metabolic activation (14, 24). The 37% Comparative mutagenicity of modified x'74 DNA survival levels, at which an average of one lethal hit per mole Data represent average values for reversion frequencies obtained in SOS- induced spheroplasts from 4 to 10 different experiments. The entries are in cule is introduced according to Poisson statistics, is attained creases above background reversion frequencies (1 x 10 ' and 10 x 10~6 for at approximately 450, 1.2, and 1.3 Â¿IMfor BPL, aBPDE, and am3 and am86. respectively). The number of lethal hits was determined from the NAAAF, respectively. Adducts formed with aBPDE and NAAAF survival data with the Poisson equation. Variations between individual experi appear to block DNA replication, and its is likely that the ments are 2- to 3-fold. Relative mutagenicities of the various treatments, however, number of lethal hits equals the number of adducts per circle vary only within 20 to 30%. Reversion frequency/10Â° lethal (4, 15). For BPL, many adducts might form per lethal event. hits In Chart 2, a representative mutagenesis experiment is de picted for the reversion of the 0x1 74 mutant am3 to wild type. TreatmentBPL Mutagenesis as a result of heat-acid-catalyzed depurination is included for comparison. The salient feature is that mutagen aBPDE 41 13 NAAAF 10 esis induced by these compounds depends on the induction of Acid-heat depurinationam35 20am8617 32 the SOS rÃ©ponse in the spheroplasts. In these studies, the SOS response was induced by exposing bacteria to UV irradia substitutions at any of the 3 positions of the (TAG) amber tion prior to their conversion to spheroplasts. In the absence of codon4 whereas am3 appears to revert exclusively at the exposure of x174DNA to the carcinogens (0 concentration), middle (adenine) position (9, 10). This discrepancy is presum there was no difference in mutagenesis between normal and ably due to the fact that am3 is located in the overlap region of SOS-induced spheroplasts. The results of a large number of genes E and D (17), whereas am86 is not located in such an experiments are averaged in Table 1 for 2 <>x174 mutants, am3 overlap region. The ability of am86 to revert at the third position and am86. To facilitate comparison, the increases in reversion is particularly important since the tested chemical carcinogens frequencies are expressed per lethal hit. In the case of heat- preferentially modify guanines (3, 21 ). Indeed, the reversion of acid, aBPDE, and NAAAF, this presumably represents one am86 occurred more frequently than did the reversion of the modification per molecule and in the case of BPL it represents am3 mutant. Reversion frequencies vary from experiment to several per molecule. DNA sequence studies of this laboratory experiment as a consequence of the varying ability of sphero- have shown that am86 is capable of reversion by single base plast preparations to express the SOS phenotype. However,

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Downloaded from cancerres.aacrjournals.org on September 24, 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 70Â°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 (â€¢)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 70Â°(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 September 24, 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-transfection 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 Â¡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

47 x 10"6 (am86). It thus appears that, for am3, depurination 1. Chen, R., Mieyal, J. J., and Goldthwait, D. A. The reaction of /3-propiolactone with derivatives of adenine and with DNA. Carcinogenesis (Lond.), 2: 73-80, through BPL is less mutagenic than when directly induced (10 x 10~6 versus 32 x 10~6). Foram86, the values are approx 1981. 2. Drinkwater, N. R., Miller, E. C., and Miller, J. A. Estimation of apurinic/ imately equal (55 X 10~6 versus 47 x 10"6). (A similar result apyrimidinic sites and phosphotri-esters in DNA treated with electrophilic carcinogens and mutagens. Biochemistry. 19: 5087-5092, 1980. was obtained in another experiment.) Since am86 is capable 3. Grunberger, D., and Weinstein, I. B. Biochemical effects of the modification of reversion at the third (guanine) position but am3 only at the of nucleic acids by certain polycyclic aromatic carcinogens. Prog. Nucleic second (adenine) position of the (TAG) amber codon, these Acid Res. Mol. Biol., 23: 105-149, 1979. 4. Hsu, W. T., Lin, E. J. S., Harvey, R. G.. and Weiss, S. B. Mechanism of data are consistent with the suggestion that direct depurination phage <>x174DNA inactivation by benzo[a]pyrene-7,8-dihydrodiol-9,1O- involves both adenine and guanine residues but that, in con epoxide. Proc. Nati. Acad. Sei. U. S. A., 74: 3335-3339, 1977. trast, mainly guanines but some adenines are removed via the 5. Kane, C. M., and Linn, S. Purification and characterization of an apurinic/ apyrimidinic endonuclease from HeLa cells. J. Biol. Chem., 256: BPL pathway. 3405-3414. 1981. In a recent study, Drinkwater et al. (2) tested a series of 6. King, H. W. S.. Osborn, M. R., and Brookes, P. The m vitro and in vivo reaction at the N7-position of guanine of the ultimate carcinogen derived chemical carcinogens for their abilities to produce apurinic from benzo[a]pyrene. Chem.-Biol. Interact., 24: 345-353, 1979. sites and their relative mutagenicities in a bacterial assay 7. Kuhnlein, U., Tsang, T. T., and Edwards, J. Characterization of DNA dam system. The compounds ranged from simple alkylating agents ages by filtration through nitrocellulose filters: a simple probe for DNA modifying agents. MutÃ¢t.Res., 64: 167-182, 1979. to the activated forms of arylamines and polycyclic hydrocar 8. Kunkel, T. A., and Loeb, L. A. On the fidelity of DNA replication. X. Effect of bons. A positive correlation was demonstrated for the ordering divalent metal ion activators and deoxyribonucleoside triphosphate pools on of the tested compounds with respect to the 2 parameters. in vitro mutagenesis. J. Biol. Chem., 254: 5718-5725, 1979. 9. Kunkel. T. A., and Loeb, L. A. On the fidelity of DNA replication. The However, the quantitative correlation between the production accuracy of Escherichia coli DNA polymerase I in copying natural DNA in of apurinic sites in vitro and the production of missense muta vitro. J. Biol. Chem., 255. 9961-9966, 1980. tions in Salmonella typhimurium was rather poor, and a direct 10. Kunkel, T. A., Schaaper, R. M., Beckman, R. A., and Loeb, L. A. On the fidelity of DNA replication. Effect of next nucleotide on proofreading. J. Biol. causal relationship was therefore considered to be unlikely. Chem., 256: 9883-9889, 1981. However, a direct comparison of depurination in vitro with 11. Lawley, P. D., and Brookes, P. Further studies on the alkylation of nucleic acids and their constituent nucleotides. Biochem. J., 89: 127-138, 1963. mutagenicity in vivo is unlikely to reflect the real relationship 12. Lindahl, T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites for the following reasons, (a) The per HIMcomparisons assume and base-excision repair. Prog. Nucleic Acid Res. Mol. Biol., 22: 135-192, the number of modifications of DNA in vitro and in vivo to be 1979. 13. Lindahl, T., and Andersson, A. Rate of chain breakage at apurinic sites in identical. In reality, the activated chemical carcinogens will double-stranded deoxyribonucleic acid. Biochemistry, 11: 3618-3623, form adducts in vivo with a variety of other compounds not 1972.

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14. Miller, J. A. Carcinogenesis by chemicals: an overviewâ€”G. H. A. Clowes 20. Schaaper, R. M., and Loeb. L. A. Oepurination is mutagenic in SOS induced Memorial Lecture. Cancer Res., 30: 559-576, 1970. cells. Proc. Nati. Acad. Sei. U. S. A., 78: 1773-1777, 1981. 15. Moore, P. A., and Strauss, B. P. Sites of inhibition of in vitro DNA synthesis 21. Segal, A., Solomon, J. J., Mignano, J., and DiÃ±o, J. The isolation and in carcinogen- and UV-treated <>x174DNA. Nature (Lond.), 278: 664-666, characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of 1979. /Â¡-propiolactone with calf thymus DNA. Chem.-Biol. Interact., 35: 349-361. 16. Osborne, M. R., Jacobs, S., Harvey, R. G., and Brookes, P. Minor products 1981. from the reaction of (+) and (â€”)benzo[a]pyrene-an//'-diolepoxide with DNA. 22. Singer, B. All oxygens in nucleic acids react with carcinogenic ethylating Carcinogenesis (Lond.), 2: 553-558, 1981. agents. Nature (Lond.), 264: 333-339, 1976. 17. Sanger, F., Coulson, A. R., Friedmann, T., Air, G. M., Barrell, B. G., Brown, 23. Singer, B., and Brent, T. P. Human lymphoblasts contain DNA-glycosylase N. L., Fiddes, J. C., Hutchinson. C. A., Ill, Slocombe. P. M., and Smith, M. activity excising N-3 and N-7 methyl and ethyl purines but not O6-alkyguan- The nucleotide sequence of bacteriophage <>x174. J. Mol. Biol., 725: ines or 1-alkyladenines. Proc. Nati. Acad. Sei. U. S. A., 78. 856-860, 1981. 225-246, 1978. 24. Sirover, M. A., and Loeb, L. A. Restriction of carcinogen-induced error 18. Sarasin, A. R., and Benoit, A. Induction of an error-prone mode of DNA incorporation during in vitro DNA synthesis. Cancer Res., 36: 516-523, repair in UV-irradiated monkey kidney cells. MutÃ¢t.Res., 70: 71 -81, 1980. 1976. 19. Sarasin, A. R., and Hanawalt, P. C. Carcinogens enhance survival of UV- 25. Stark, A. A., Essigman, J. M., Demain, A. L., Skopek, T. R., and Wogan, G. irradiated simian virus 40 in treated monkey kidney cells. Induction of a N. Aflatoxin B. mutagenesis, DNA binding and adduci formation in Salmo- recovery pathway? Proc. Nati. Acad. Sei. U. S. A., 75: 346-350. 1978. nella typhimurium. Proc. Nati. Acad. Sci. U. S. A., 76: 1343-1347, 1979.

SEPTEMBER 1982 3485

Roeland M. Schaaper, Barry W. Glickman and Lawrence A. Loeb

Cancer Res 1982;42:3480-3485.

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