Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1378-1382, April 1977 Biochemistry
A bacteriophage system for screening and study of biologically active polycyclic aromatic hydrocarbons and related compounds (benz[aJanthracene and benzolalpyrene derivatives/Escherichia coli spheroplasts/chemical carcinogenesis/infectious nucleic acids)
WEN-TAH Hsu*, RONALD G. HARVEYt, EDWARD J. S. LIN*, AND SAMUEL B. WEISS * *The Franklin McLean Memorial Research Institute, the Departments of Biochemistry and Microbiology, and tThe Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637
Communicated by Leon 0. Jacobson, December 27,1976
ABSIRACT The usefulness of bacterial viruses for detecting substances that are potentially carcinogenic is reexamined as a model system for screening biologically active polycyclic aromatic hydrocarbons. A modification of the original assay procedure allows one to distinguish between aromatics that can modify the biological activity of infectious nucleic acids directly and those polycyclic aromatic hydrocarbons that require metabolic activation by Escherichia coli enzymes. The effect of chemical modification of several different polycyclic aromatic hydrocarbons, with respect to their biological activity in the phage assay system, is described. Among the 31 different compounds examined, (anti-benzo apyrene-7,8-diol-9,1O-epoxide was the most potent inhibitor of infectious phage nucleic acid. The (+) and (-) isomers of the above racemic mixture did not differ significantly in their capacity to inhibit phage replica-
QB virus was obtained from Drs. A. Palmenberg and P. Kaesberg (The University of Wisconsin). Assay for Infectious Nucleic Acids. When intact QB RNA or OX174 DNA is mixed with E. coli spheroplasts, the nucleic acid is adsorbed by the host and initiates the production of new virions. After incubation of the infected spheroplasts for 5-10 min at 250, the infection mixture is plated with cells of a sensitive strain of E. coli (indicator) onto agar plates so that the formation of new viral plaques can be measured. Plaques become visible within 6-10 hr after incubation of the plates at 370 and 31° for QB and OX174 phage, respectively. To assess the effect of PAH on virus replication, we used three procedures that differ in the sequential treatment of viral nucleic acid with PAH and/or spheroplasts and in the processing of the test nucleic acid for determination of infectivity. Method A. In this procedure, infectious nucleic acid and the test compound were mixed for a few minutes at room temperature and then added to a suspension of E. coli spheroplasts. After 5-10 min of incubation at 250, 3 ml of soft agar and two drops of a suspension of indicator E. coli were added, and the entire mixture was plated and incubated as described above. Modified Method A. Immediately before use, E. coli spheroplasts were first infected with the test infectious nucleic acid by mixing of equal volumes of both and kept at 40. Aliquots of infected spheroplasts were then mixed with PAH and incubated at 25° for 5-10 min before plating. Method B. In this procedure, referred to as the "pretreatment" method, infectious nucleic acid and hydrocarbon were mixed together and incubated at 370 for 10 min. Twenty volumes of ethanol were added, and the mixture was kept at -200 overnight. The precipitate that formed was collected by centrifugation and washed once with acetone. The pellet was resuspended in water, and the nucleic acid concentration was determined by its absorbance at 260 nm. When the nucleic acid solution had been diluted, aliquots were mixed with E. coli spheroplasts and assayed for their plaque-forming capability (infectivity) as described for method A. The effectiveness of the above procedure for removing unreacted PAH from nucleic acid is discussed below. Synthesis and Storage of Hydrocarbons. The hydrocarbons
tion.
Studies done in this laboratory more than a decade ago indicated the usefulness of bacterial viruses in the detection of substances that were potentially carcinogenic (1, 2). In these studies we used the incubation of Escherichia coli spheroplasts with infectious nucleic acid, prepared either from MS2 or 4X174 bacteriophages, and determined the extent of inhibition of viral replication after addition of polycyclic aromatic hydrocarbons (PAH). A high correlation between carcinogenic activity in rats and inhibition of viral replication was observed with different hydrocarbons. These findings were confirmed by another laboratory using this same system (3, 4). The present report is an extension of our earlier work in which biologically reactive hydrocarbons are identified in an infectious nucleic acid-spheroplast assay system. By applying a simple modification of the original assay procedure, it is possible to distinguish between aromatics that can inactivate infectious nucleic acids directly and those that require metabolic activation by E. coli spheroplasts. METHODS Preparation of Spheroplasts and Infectious Nucleic Acid. Spheroplasts were prepared by treatment of E. coil strain K12W1485 with lysozyme according to a method described (1). For QB phage production and phenol extraction of the viral RNA, the method of Eoyang and August (5) was used. The bacteriophage OX174am3 and its sensitive host, E. coil HF4714, were the gifts of Dr. L. B. Dumas (Northwestern University).
benz[a]anthracene (BA), benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (Me2BA), and phenanthrene were purchased from commercial sources and were purified by recrystallization as necessary to give a single spot on thin-layer chromatography. The K-region oxides, phenanthrene-9,10oxide, Me2BA-5,6-oxide, BP-4,5-oxide, 1-methylphenanthrene-9,10-oxide, and benzo[c]phenanthrene-9,10-oxide, and the K-region dihydrodiols, trans-5,6-dihydroxy-5,6-dihydroMe2BA (Me2BA-5,6-diol) and trans-7,8-dihydroxy-7,8-dihydro-BP (BP-4,5-diol), were synthesized by procedures described
Abbreviations: PAH, polycyclic aromatic hydrocarbons; BA, benz[a ]anthracene; Me2BA, 7,12-dimethyl-BA; MeBA, methyl-BA; BP, benzo[alpyrene; H4BP, 7,8,9,10-tetrahydro-BP; anti-BP-diolepoxide, (+)trans-7,8-dihydroxy-anti-9,10-epoxy-H4BP; syn-BP-diolepoxide, (+)-trans-7,8-dihydroxy-syn-9,10-epoxy-H4BP; BP-4,5-diol, trans4,5-dihydroxy-4,5-dihydro-BP; BP-7,8-diol, trans-7,8-dihydroxy7,8-dihydro-BP; Me2BA-5,6-diol, trans-5,6-dihydroxy-5,6-dihydroMe2BA.
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(6-8). (4)-trans-7,8-Dihydroxy-anti-9,10-epoxy-7,89,;10tetrahydro-BP (anti-BP-diolepoxide) and (±)-trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydro-BP (syn-BPdiolepoxide) and trans-7,8-dihydroxy-7,8-dihydro-BP (BP7,8-diol) were also prepared by published methods (9, 10). The corresponding anti- and syn-BA-diolepoxides were obtained through a modification of the same methods (R. G. Harvey and F. A. Beland, unpublished data). The phenols 4-,7-, and 8hydroxy-BP were synthesized by procedures of R. G. Harvey and P. Fu (unpublished data). The following compounds were synthesized by the method given in the reference cited in parentheses: 7-oxiranyl-12-MeBA (11); Me2BA-5,6-dione (6, 7); 7-hydroxymethyl-12-MeBA-5,6-oxide (12); 1,4-methyl-2phenylnaphthalene-3,2'-dicarboxaldehyde (13); 7-bromomethyl-12-MeBA (14); 7-hydroxymethyl-12-MeBA (15); 7,12-diformyl-BA (14); 7-methyl-12-formyl-BA (15); 7-formyl-12-!MeBA(14); and BP-4,5-dione (7, 8). The epoxides, 7,8-epoxy-H4BP and 9,10-epoxy-H4BP, were synthesized according to a procedure to be described elsewhere (R. G. Harvey, unpublished data). The method for the resolution of (+)-antiBP-diolepoxide into (+) and (-) isomers has been submitted for publication (H. Cho and R. G. Harvey). All compounds were of the highest attainable purity (>95%); nuclear magnetic resonance spectra of each compound were consistent with the assigned structure. Most of the compounds gave single spots on silica gel plates. Stock solutions of the hydrocarbons were made up in dimethylformamide, in tubes wrapped with aluminum foil, and stored at 4°. For the experiments described in this study, only hydrocarbon-dimethylformamide solutions less than 2 weeks old were used, even though no loss in biological activity was observed over a period of 2 months for the highly reactive isomers of BP-diolepoxide. RESULTS Table 1 shows the results obtained when various compounds were mixed with QB RNA, and after the introduction of E. coli spheroplasts, the mixture was plated for plaque development. Whereas BA and phenanthrene oxide were virtually inactive at each of the concentrations examined, the remaining aromatics exhibited various degrees of inhibition of plaque formation. BP, its K-region oxide, and Me2BA are active only at relatively high concentrations. The two diastereoisomeric BP-7,8-diol-9,10-epoxides were inhibitory even at the lowest concentration (0.1 Mg/ml). The relative configuration of the epoxide and diol groups is apparently important in this assay system; the anti-isomer exhibits a greater inhibitory action than the syn-form at the lower concentrations. In our earlier studies, only a limited number of aromatics were tested in the phage assay system. Since some difficulty in reproducibility was found with infectious RNA, we turned to cX174 DNA as the infectious agent. Table 2 summarizes the results obtained with 31 different compounds in several separate experiments. A similar activity pattern was detected among those compounds tested with both QB RNA and 4X174 DNA as the infectious agents. The results in Table 2 suggest the following: when the 7-methyl group of Me2BA is brominated or substituted by an oxiranyl group, the inhibitory phenomenon is strongly enhanced, the latter modification being more effective for inhibition than the former. The replacement of one or both methyl groups of Me2BA with formyl groups, or that Af the 7-methyl group with a hydroxymethyl group, results in in appreciable reduction of the potency of Me2BA. Whereas :he presence of quinone or hydroxyl groups at the 5,6-position K-region) of Me2BA lowers the inhibitory response, the introduction of a 5,6-oxide function markedly stimulates inhi-
Proc. Natl. Acad. Sci. USA 74 (1977)
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Table 1. Effect of PAH and their derivatives on the infectivity of QB RNA (method A) Concentration
Compound None BA
Me2 BA Me2 BA-5,6-oxide
Infec% Inhibition
(g/ml)
tivity (PFU)
0 0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0
310 327 454 247 285 129 5 347 296 70
0 0 0 20 8 58 99 0 6 77
0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0
272 306 230 306 263 91 352 274 59
12 1 26 1 15 71 0 12 81
0.1 1.0 10.0
171 2 0
45 99 >99
0.1 1.0 10.0
183 56 0
41 82 >99
Phenanthrene-
9,10-oxide BP
BP-4,5-oxide anti-BP-diolepoxide
syn-BP-diolepoxide
QB RNA (0.01 ,g) and a given PAH, in the amounts shown above, combined in a total volume of 0.1 ml containing 2% dimethylformamide. To this mixture was added an equal volume of E. coli spheroplasts (method A). After 5 min at 250, this mixture was plated on agar with E. coli Q13 as indicator and placed in an incubator at 370 overnight. The plaque values (PFU = plaque-forming units) shown represent an average for two plates. The concentration (,ug/ml) represents the amount of PAH present in 1 ml of the final infection were
mixture.
bition. The opening of the K-region of Me2BA results in the loss of its biological activity. Simultaneous trans-hydroxylation at C-8,9 and epoxidation at the C-9,10 in the anti-configuration resulted in a pronounced increase in the activity of BA, whereas a similar oxidative transformation to the syn-isomer had no effect on the inhibitory response. Table 2 also indicates that introduction of hydroxyl groups at the 4, 7, or 8 position of BP has little or no effect on the biological activity of kX174 DNA. Whereas the addition of quinone or of hydroxyl groups to the 4,5 positions fails to show any significant influence on biological response, the presence of an epoxide group across these positions results in a marked increase in the activity of BP. Trans-hydroxylation at the 7,8 positions of BP produces further enhancement of activity. Introduction of an epoxide function to various positions of BP had variable effects. Thus, whereas an epoxide group at the K-region or the 9,10 positions resulted in little or no change in activity, the 7,8-epoxy derivative was strongly inhibitory. As was observed with QB RNA, the 7,8-diol-9,10-epoxide derivatives of BP are also highly inhibitory, the anti-isomer being the most active compound so far tested. Phenanthrene and its three oxide derivatives were noninhibitory. Fig. 1 illustrates the dose response of OXX174 DNA to the two diolepoxide isomers of BP and BA when assayed by modified
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Proc. Natl. Acad. Sci. USA 74 (1977)
Table 2. Effect of PAH derivatives on infectivity of OX174 DNA (modified method A) % Inhibition
Compound Phenanthrene Phenanthrene-9,10-oxide 1-Methylphenanthrene9,10-oxide Benzo [c I phenanthrene9,10-oxide BA Me2BA 7-Bromomethyl-12-MeBA
7-Hydroxymethyl-12-MeBA 7,12-Diformyl-BA 7-Formyl-12-MeBA 12-Formyl-7-MeBA Me2 BA-5,6-dione Me2 BA-5,6-diol 7-Oxiranyl-12-MeBA 1,4-Dimethyl-2-phenylnaphthalene-3,2' -dicarboxaldehyde 7-Hydroxymethyl-12MeBA-5,6-oxide BA-5,6-oxide anti-BA-diolepoxide
syn-BA-diolepoxide BP 4-HO-BP
7-HO-BP 8-HO-BP BP-4,5-dione BP-4,5-dlol
BP-7,8-diol BP-4,5-oxide 7,8-Epoxy-H4BP
9,10-Epoxy-H4BP anti-BP-diolepoxide syn-BP-diolepoxide
at 0.2 mg/ml
0 0
100
at 2.0 ,4g/ml
7
anti - BA- diolepoxideo
0 0 0 3 28 72 82 29 20 35 52 44 8 >99
10
24
12 43 44 0 14 12 28 0 0 0 40 24 48 23 95 35
70 >99 97 12 54 9 41 20 0 7 92 61 >99 35 >99 77
The assay was similar to that described in the footnote for Table 1, except that 4X174am3 DNA was used as the test nucleic acid and E. coli spheroplasts were preinfected with this DNA. Two-tenths milliliter of the infected spheroplasts was introduced into tubes containing PAH in 4 jil of dimethylformamide; after 5 min at 250, the spheroplasts were plated with E. coli HF4714 as the indicator (modified method A). For the preinfection, equal volumes of E. coli spheroplasts and OX174 DNA were mixed in 5 mM EDTA and kept at 40 until used. The concentration of the ihfecting DNA was approximately 0.03 jg/ml, and 0.2 ml of infected spheroplasts yielded between 100 and 300 plaques in the absence of PAH after plating. Percent inhibition was calculated from the average number of plaques observed from triplicate plates.
method A. The concentrations of BP-diolepoxide required for 50% inactivation of infectivity (150) are approximately 0.04 ,ug/ml (0.13 ,AM) and 0.6 Ag/ml (2 ,uM) for the anti- and synforms, respectively. The I'50 molar concentrations are probably lower than the values derived from this experiment, since stability studies with each isomer in aqueous media (data not shown) indicate that both isomers are unstable, the syn-form being less stable than the anti-form. The I50 for the anti-form of the BA-diolepoxide derivative was approximately 2 ,uM, whereas the syn-BA-diolepoxide demonstrated no significant inhibition. The stability of the BA-derivatives in aqueous solution was not examined.
syn-BP-diolepoxide
o C
5 19 36 0 0 4 13 10 0 52
0
.~50
C
syn- BA - diolepoxide
0
0.5
1.0
1.5
2.0
ing of PA H per m u FIG. 1. Effect of the diolepoxides of BP and BA on the infectivity of OX174 DNA. The assay for infectivity was done by modified method A. The concentrations of the anti- and syn-forms of 7,8dihydroxy-9,10-epoxy-BP and 8,9-dihydroxy-10,1t-epoxy-BA in the E. coli spheroplast mixture (0.2 ml) were as indicated. The diolepoxides were the (+) racemic mixtures. Three nanograms of DNA from the amber phageoX174am3 were used as infectious nucleic acid in the same reaction mixture. Each point represents values calculated from the average of three plates.
Using the same assay procedure (modified method A), Fig. 2 shows that the separated (-) and (+) stereoisomers of antiBP-diolepoxide exhibit essentially the same inhibitory dose response as the racemic mixture. In order to distinguish between active aromatics that are
capable of reacting directly with nucleic acid molecules and those that are inactive but can be activated by intracellular modification, we devised a nucleic acid "pretreatment" procedure (method B). In this procedure, the infectious nucleic acid
1oo0 4.. C
C
al
.' 50
0.5 szg of anti - BP - diolepoxide per ml
FIG. 2. Effect of the (-) and (+) isomers of anti-BP-diolepoxide on the infectivity of OX174 DNA. The assay procedure and conditions of infection were the same as in the legend of Fig. 1 except that the separated (-) and (+) isomers of anti-7,8-dihydroxy-9,10-epoxy-BP were used, at the concentrations shown above, in reaction mixtures with OX174am3 DNA.
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Table 3. Effect of OX174 DNA pretreatment with PAH derivatives on infectivity Compound
None BA Me2BA 7-Bromomethyl-12-MeBA 12-Formyl-7-MeBA 7-Oxiranyl-12-MeBA 7-Hydroxymethyl-12-MeBA5,6-oxide Me2 BA-5,6-oxide anti-BA-diolepoxide
syn-BA-diolepoxide BP BP-7,8-diol
7,8-Epoxy-H, BP 9,10-Epoxy-H4BP anti-BP-diolepoxide syn-BP-diolepoxide
Phage titer (PFU)
% Inhibition
108 100 43 110 41
0 0 7 60 0 62
115 20 24 100 99 80 107 111 0 60
0 81 78 7 8 26 0 0 >99 46
105
0
0.05
0.10
0.15
0.20
pg of anti-BP-diolepoxide
,X174am3 DNA (10 Mg) and 0.5 ig of PAH were mixed in a final volume of 0.1 ml and allowed to stand at 370 for 10 min. The DNA was reisolated after treatment with solvents as described under method B in Methods, and assayed for infectivity. Recovery of DNA after solvent treatment was nearly quantitative.
FIG. 3. Effect on infectivity of QB RNA pretreated with different concentrations of anti-BP-diolepoxide. QB RNA (10 Mg) was mixed with varying concentrations of anti-BP-diolepoxide in a final volume of 0.05 ml containing 0.01 M Tris-HCl (pH 7.5) and 4% dimethylformamide. After 10 min at 370, the unreacted hydrocarbon was removed by ethanol precipitation and acetone washing (method B). QB RNA was dissolved in 1 ml of water, its concentration was determined, and, after appropriate dilution, it was assayed in triplicate for infectivity with spheroplasts.
is preincubated with PAH and unreacted hydrocarbon is removed by solvent treatment prior to spheroplast exposure. Table 3 shows that when 4X174 DNA was pretreated with BA, Me2BA, 12-formyl-7-MeBA, 7-hydroxymethyl-12-MeBA5,6-oxide, syn-BA-diolepoxide, BP, BP-7,8-diol, 7,8-epoxyH4BP, and 9,10-epoxy-H4BP and then assayed after removal of the unreacted compounds, little, if any loss of infectivity occurred. These results suggest that none of the above compounds is capable of inactivating infectious nucleic acids unless it is converted to some chemically reactive form after incorporation into spheroplasts, as was demonstrated in the results of Table 2. Exceptions are BA, syn.BA-diolepoxide and 9,10epoxy-H4BP which are relatively inactive even when assayed by the modified method A procedure (Table 2). The remaining compounds listed in Table 3 are effective inhibitors of OXX174 DNA expression when they are assayed by the pretreatment procedure; hence, these compounds are capable of interacting with and modifying nucleic acid molecules directly without intracellular activation. Similar results, with the same compounds, were found when QB RNA served as the infectious agent. It is important to note that, in control experiments, when E. coli tRNA was pretreated with anti-BP-diolepoxide and extracted by the solvent treatment procedure used in the above assay, no residual inhibitory activity could be detected in the recovered RNA fraction upon the addition of test infectious nucleic acid. Fig. 3 shows the effect of increasing concentration of antiBP-diolepoxide on the expression of QB RNA as determined by the pretreatment procedure. For 10 ,gg of the viral RNA, almost complete loss of infectivity (97%) was achieved by exposure to 0.1 jig of the diolepoxide, and 50% inhibition occurred at a molar ratio of RNA nucleotide to BP-diolepoxide of approximately 300:1. DISCUSSION Recently, it has been shown that certain derivatives of PAH can react covalently with nucleic acids, in vivo and in vitro, providing a possible explanation for the way in which these chemical agents elicit biological responses in animal and mi-
crobial organisms (16-18). In this report, we have reexamined the application of a phage assay system, previously described by this laboratory (1), for the detection of biologically active aromatics. The results described here are consistent with our earlier findings and indicate that certain compounds, known to be carcinogenic in animals, are also capable of blocking the replication of infectious nucleic acids in E. coli spheroplasts. The phage nucleic acid may be either RNA or DNA, and the assay system is suitable for quantitating the relationship between dose and biological response for various active hydrocarbons. Repeat experiments indicate that, although the plaque titer assay is highly reproducible, differences of 20% are not significant. Pretreatment of the infectious nucleic acid with the hydrocarbon derivatives and then removal of unreacted compound prior to assay allows one to discriminate between those aromatics that can inactivate infectious nucleic acids directly and those that require metabolic activation to demonstrate an inhibitory response. Presumably, direct inactivation of infectivity occurs with aromatics that are capable of binding nucleic acid molecules without any chemical alteration, whereas aromatics that require exposure to spheroplasts become reactive only after intracellular modification. In general, those aromatics that exhibit the highest activity in the assay procedures described here are those known to function as alkylating agents capable of direct covalent interaction with nucleic acids. These include the K-region oxides and the non-K-region diolepoxides, both of which have been shown to be metabolites of the parent hydrocarbon formed by microsomal enzymes (17-21). Neither 7,8-epoxy-H4BP nor 9,10-epoxy-H4BP is apparently capable of modifying 4X174 DNA directly. The former derivative, however, can be converted into an active form by E. coli spheroplasts. These results either indicate the importance of the position of the epoxide group or reflect the instability of the 9,10-epoxy-H4BP. The other particularly active compounds are the 7-bromomethyl and 7-oxiranyl derivatives of 12-MeBA; these are not metabolic products, but are expected, on chemical grounds, to be effective
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alkylating agents. The former derivative has been shown to react with nucleic acids (22), and both compounds are known to be carcinogenic (22, 23). Recently, in the process of BP-induced carcinogenesis, BP-diolepoxide has been implicated as the ultimate carcinogen since it appears as a conjugate of nucleic acids when BP is incubated with cells in culture (19-21, 24-27). Osborne et al. (26) found that both anti- and syn-BP-diolepoxide isomers react readily with DNA, yielding similar products. Although preliminary data indicate its carcinogenicity to be weak (S. Hecht, unpublished data), presumably due to its instability, the antiBP-diolepoxide is the most active mutagenic derivative synthesized thus far (19, 28). Our experiments are in general agreement with the above observations. The two stereoisomers of BP-7,8-diol-9,10-epoxide react directly with QB RNA and (DX174 DNA to inactivate both nucleic acids; the anti-form is the most effective inhibitor of infectivity examined in this study. Under the conditions used for the pretreatment procedure, approximately 14 molecules of diolepoxide are required for each molecule of QB RNA if 50% inactivation is to be achieved (Fig. 3). The studies with the two isomers of BA-diolepoxide show that the anti-form has a strong inhibitory effect on viral replication, whereas the synform has little or no effect. These results suggest that the diolepoxide derivatives of BP and BA are highly active as inhibitors of the replication of infectious nucleic acid; the geometric configuration of these isomers appears to be an important determinant for the degree of biological activity expressed. The (-) and (+) isomers of anti-BP-diolepoxide showed little difference in their inhibitory response. It is possible that some of the results obtained with these highly reactive compounds also reflect relative differences in their stability. Recently, microorganisms and cultured animal cells have been adopted, with increasing frequency, for the detection and identification of toxic chemical substances (29). The use of biologically active nucleic acids, in the manner described here, may serve a similar purpose. Because of the rapidity and economy of evaluating the hydrocarbon response, the phage assay system is well suited for the monitoring of large numbers of compounds. It also has an advantage for detecting highly reactive, but unstable, derivatives of PAH, which might go undetected in other test systems yet be important intermediates in the expression of PAH toxicity. In addition to certain obvious differences between the assay system reported here and the Salmonella typhimurium-mutagen system (30), there appears to be a difference in the response to specific hydrocarbons. For example, BP and Me2BA are not mutagenic in the Salmonella reversion test unless there is prior activation by mammalian enzymes (31); however, these same compounds are inhibitory for phage replication in E. coli spheroplasts. Schumm (3) and Fried (4) identified various oxidation products of labeled Me2BA after its incubation with E. coli spheroplasts. The disparity between E. coli and S. typhimurium in their response to certain hydrocarbons may reflect metabolic differences for these two organisms, or may result from the altered permeability properties of lysozyme-formed E. coli spheroplasts used in this study. Nonetheless, the preincubation of hydrocarbons with mammalian extracts, as well as the use of certain bacterial mutants deficient in DNA excision repair (32), may improve the sensitivity and broaden the scope for detecting potentially harmful substances in the infectious nucleic acid-spheroplast system. The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the United States Energy Research and
Proc. Natl. Acad. Sci. USA 74 (1977) Development Administration under Contract no. EY-76-C-02-0069. This study was partly supported by a grant from the American Cancer Society (BC-132A) and by funds from EPA Subagreement no. 77BAN. We thank Drs. H. Cho and K. B. Sukumaran and Ms. Cecilia Cortez for furnishing samples of some of the compounds used. 1. Hsu, W. T., Moohr, J. W. & Weiss, S. B. (1965) Proc. Natl. Acad. Sci. USA 53,517-524. 2. Hsu, W. T., Moohr, J. W., Tsai, A. Y. M. & Weiss, S. B. (1966) Proc. Natl. Acad. Sci. USA 55, 1475-1482. 3. Schumm, D. L. (1969) Ph.D. Dissertation, University of Chicago. 4. Fried, J. (1974) in Chemical Carcinogenesis, eds. T'so, P. 0. P. & di Paolo, J. A. (Dekker, New York), pp. 197-215. 5. Eoyang, L. & August, T. P. (1968) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. 12, Part B, pp. 530-540. 6. Goh, S. H. & Harvey, R. G. (1973) J. Am. Chem. Soc. 95,242243. 7. Harvey, R. G., Goh, S. H. & Cortez, C. (1975) J. Am. Chem. Soc. 97,3468-3479. 8. Cho, H. & Harvey, R. G. (1974) Tetrahedron Lett., 14911494. 9. Beland, F. A. & Harvey, R. G. (1976) J. Chem. Soc. Chem. Commun., 84-85. 10. McCaustland, D. J. & Engel, J. F. (1975) Tetrahedron Lett., 2549-2552. 11. Harvey, R. G., Pataki, J., Wilke, R. N., Flesher, J. W. & Soedigdo, S. (1976) Cancer Lett. 1, 339-344. 12. Sims, P. (1973) Biochem. J. 131, 405-413. 13. Hadler, H. I. & Kryger, A. C. (1960) J. Org. Chem. 25, 18961901. 14. Pataki, J., Wlos, R. & Cho, Y. (1968) J. Medic. Chem. 11, 1083-1086. 15. Boyland, E. & Sims, P. (1965) Biochem. J. 95,780-787. 16. Miller, E. C. & Miller, J. A. (1974) in The Molecular Biology of Cancer, ed. Busch, H. (Academic Press, New York), pp. 377402. 17. Sims, P. & Grover, P. L. (1974) Adv. Cancer Res. 20, 165-274. 18. Heidelberger, C. (1975) Annu. Rev. Biochem. 44,79-121. 19. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 20. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 21. Yang, S. K., McCourt, D. W., Roller, P. P. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73,2594-2598. 22. Dipple, A., Brookes, P., MacKintosh, D. S. & Rayman, M. P. (1971) Biochemistry 10,4323-4330. 23. Flesher, J. W., Harvey, R. G. & Sydnor, K. (1976) Int. J. Cancer 18,351-353. 24. Daudel, P., Deuquesne, M., Vigny, P., Grover, P. L. & Sims, P. (1975) FEBS Lett. 57, 250-253. 25. Ivanovic, V., Geacintov, N. E. & Weinstein, I. B. (1976) Biochem. Biophys. Res. Commun. 70, 1172-1179. 26. Osborne, M. R., Thompson, M. H., Tarmy, E. M., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Chem. Biol. Interact. 14, 343-348. 27. Weinstein, I. B, Jefferey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 28. Newbold, R. F. & Brookes, P. (1976) Nature 261,52-54. 29. Montesano, R., Bartsh, H. & Tomatis, L., eds. (1976) Screening Test in Chemical Carcinogenesis (International Agency for Research on Cancer, World Health Organization, Lyon), IARC Scientific Publication no. 12. 30. McCann, J., Choi, E., Yamasaki, E. & Ames, B. N. (1975) Proc. Natl. Acad. Sci. USA 72,5135-5139. 31. Ames, B. N., Durston, W. E., Yamasaki, E. & Lee, F. D. (1973) Proc. Natl. Acad. Sci. USA 70,2281-2285. 32. Ames, B. N., Lee, F. D. & Durston, W. E. (1973) Proc. Natl. Acad. Sci. USA 70, 782-786.
A bacteriophage system for screening and study of biologically active polycyclic aromatic hydrocarbons and related compounds (benz[aJanthracene and benzolalpyrene derivatives/Escherichia coli spheroplasts/chemical carcinogenesis/infectious nucleic acids)
WEN-TAH Hsu*, RONALD G. HARVEYt, EDWARD J. S. LIN*, AND SAMUEL B. WEISS * *The Franklin McLean Memorial Research Institute, the Departments of Biochemistry and Microbiology, and tThe Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, Illinois 60637
Communicated by Leon 0. Jacobson, December 27,1976
ABSIRACT The usefulness of bacterial viruses for detecting substances that are potentially carcinogenic is reexamined as a model system for screening biologically active polycyclic aromatic hydrocarbons. A modification of the original assay procedure allows one to distinguish between aromatics that can modify the biological activity of infectious nucleic acids directly and those polycyclic aromatic hydrocarbons that require metabolic activation by Escherichia coli enzymes. The effect of chemical modification of several different polycyclic aromatic hydrocarbons, with respect to their biological activity in the phage assay system, is described. Among the 31 different compounds examined, (anti-benzo apyrene-7,8-diol-9,1O-epoxide was the most potent inhibitor of infectious phage nucleic acid. The (+) and (-) isomers of the above racemic mixture did not differ significantly in their capacity to inhibit phage replica-
QB virus was obtained from Drs. A. Palmenberg and P. Kaesberg (The University of Wisconsin). Assay for Infectious Nucleic Acids. When intact QB RNA or OX174 DNA is mixed with E. coli spheroplasts, the nucleic acid is adsorbed by the host and initiates the production of new virions. After incubation of the infected spheroplasts for 5-10 min at 250, the infection mixture is plated with cells of a sensitive strain of E. coli (indicator) onto agar plates so that the formation of new viral plaques can be measured. Plaques become visible within 6-10 hr after incubation of the plates at 370 and 31° for QB and OX174 phage, respectively. To assess the effect of PAH on virus replication, we used three procedures that differ in the sequential treatment of viral nucleic acid with PAH and/or spheroplasts and in the processing of the test nucleic acid for determination of infectivity. Method A. In this procedure, infectious nucleic acid and the test compound were mixed for a few minutes at room temperature and then added to a suspension of E. coli spheroplasts. After 5-10 min of incubation at 250, 3 ml of soft agar and two drops of a suspension of indicator E. coli were added, and the entire mixture was plated and incubated as described above. Modified Method A. Immediately before use, E. coli spheroplasts were first infected with the test infectious nucleic acid by mixing of equal volumes of both and kept at 40. Aliquots of infected spheroplasts were then mixed with PAH and incubated at 25° for 5-10 min before plating. Method B. In this procedure, referred to as the "pretreatment" method, infectious nucleic acid and hydrocarbon were mixed together and incubated at 370 for 10 min. Twenty volumes of ethanol were added, and the mixture was kept at -200 overnight. The precipitate that formed was collected by centrifugation and washed once with acetone. The pellet was resuspended in water, and the nucleic acid concentration was determined by its absorbance at 260 nm. When the nucleic acid solution had been diluted, aliquots were mixed with E. coli spheroplasts and assayed for their plaque-forming capability (infectivity) as described for method A. The effectiveness of the above procedure for removing unreacted PAH from nucleic acid is discussed below. Synthesis and Storage of Hydrocarbons. The hydrocarbons
tion.
Studies done in this laboratory more than a decade ago indicated the usefulness of bacterial viruses in the detection of substances that were potentially carcinogenic (1, 2). In these studies we used the incubation of Escherichia coli spheroplasts with infectious nucleic acid, prepared either from MS2 or 4X174 bacteriophages, and determined the extent of inhibition of viral replication after addition of polycyclic aromatic hydrocarbons (PAH). A high correlation between carcinogenic activity in rats and inhibition of viral replication was observed with different hydrocarbons. These findings were confirmed by another laboratory using this same system (3, 4). The present report is an extension of our earlier work in which biologically reactive hydrocarbons are identified in an infectious nucleic acid-spheroplast assay system. By applying a simple modification of the original assay procedure, it is possible to distinguish between aromatics that can inactivate infectious nucleic acids directly and those that require metabolic activation by E. coli spheroplasts. METHODS Preparation of Spheroplasts and Infectious Nucleic Acid. Spheroplasts were prepared by treatment of E. coil strain K12W1485 with lysozyme according to a method described (1). For QB phage production and phenol extraction of the viral RNA, the method of Eoyang and August (5) was used. The bacteriophage OX174am3 and its sensitive host, E. coil HF4714, were the gifts of Dr. L. B. Dumas (Northwestern University).
benz[a]anthracene (BA), benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (Me2BA), and phenanthrene were purchased from commercial sources and were purified by recrystallization as necessary to give a single spot on thin-layer chromatography. The K-region oxides, phenanthrene-9,10oxide, Me2BA-5,6-oxide, BP-4,5-oxide, 1-methylphenanthrene-9,10-oxide, and benzo[c]phenanthrene-9,10-oxide, and the K-region dihydrodiols, trans-5,6-dihydroxy-5,6-dihydroMe2BA (Me2BA-5,6-diol) and trans-7,8-dihydroxy-7,8-dihydro-BP (BP-4,5-diol), were synthesized by procedures described
Abbreviations: PAH, polycyclic aromatic hydrocarbons; BA, benz[a ]anthracene; Me2BA, 7,12-dimethyl-BA; MeBA, methyl-BA; BP, benzo[alpyrene; H4BP, 7,8,9,10-tetrahydro-BP; anti-BP-diolepoxide, (+)trans-7,8-dihydroxy-anti-9,10-epoxy-H4BP; syn-BP-diolepoxide, (+)-trans-7,8-dihydroxy-syn-9,10-epoxy-H4BP; BP-4,5-diol, trans4,5-dihydroxy-4,5-dihydro-BP; BP-7,8-diol, trans-7,8-dihydroxy7,8-dihydro-BP; Me2BA-5,6-diol, trans-5,6-dihydroxy-5,6-dihydroMe2BA.
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(6-8). (4)-trans-7,8-Dihydroxy-anti-9,10-epoxy-7,89,;10tetrahydro-BP (anti-BP-diolepoxide) and (±)-trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydro-BP (syn-BPdiolepoxide) and trans-7,8-dihydroxy-7,8-dihydro-BP (BP7,8-diol) were also prepared by published methods (9, 10). The corresponding anti- and syn-BA-diolepoxides were obtained through a modification of the same methods (R. G. Harvey and F. A. Beland, unpublished data). The phenols 4-,7-, and 8hydroxy-BP were synthesized by procedures of R. G. Harvey and P. Fu (unpublished data). The following compounds were synthesized by the method given in the reference cited in parentheses: 7-oxiranyl-12-MeBA (11); Me2BA-5,6-dione (6, 7); 7-hydroxymethyl-12-MeBA-5,6-oxide (12); 1,4-methyl-2phenylnaphthalene-3,2'-dicarboxaldehyde (13); 7-bromomethyl-12-MeBA (14); 7-hydroxymethyl-12-MeBA (15); 7,12-diformyl-BA (14); 7-methyl-12-formyl-BA (15); 7-formyl-12-!MeBA(14); and BP-4,5-dione (7, 8). The epoxides, 7,8-epoxy-H4BP and 9,10-epoxy-H4BP, were synthesized according to a procedure to be described elsewhere (R. G. Harvey, unpublished data). The method for the resolution of (+)-antiBP-diolepoxide into (+) and (-) isomers has been submitted for publication (H. Cho and R. G. Harvey). All compounds were of the highest attainable purity (>95%); nuclear magnetic resonance spectra of each compound were consistent with the assigned structure. Most of the compounds gave single spots on silica gel plates. Stock solutions of the hydrocarbons were made up in dimethylformamide, in tubes wrapped with aluminum foil, and stored at 4°. For the experiments described in this study, only hydrocarbon-dimethylformamide solutions less than 2 weeks old were used, even though no loss in biological activity was observed over a period of 2 months for the highly reactive isomers of BP-diolepoxide. RESULTS Table 1 shows the results obtained when various compounds were mixed with QB RNA, and after the introduction of E. coli spheroplasts, the mixture was plated for plaque development. Whereas BA and phenanthrene oxide were virtually inactive at each of the concentrations examined, the remaining aromatics exhibited various degrees of inhibition of plaque formation. BP, its K-region oxide, and Me2BA are active only at relatively high concentrations. The two diastereoisomeric BP-7,8-diol-9,10-epoxides were inhibitory even at the lowest concentration (0.1 Mg/ml). The relative configuration of the epoxide and diol groups is apparently important in this assay system; the anti-isomer exhibits a greater inhibitory action than the syn-form at the lower concentrations. In our earlier studies, only a limited number of aromatics were tested in the phage assay system. Since some difficulty in reproducibility was found with infectious RNA, we turned to cX174 DNA as the infectious agent. Table 2 summarizes the results obtained with 31 different compounds in several separate experiments. A similar activity pattern was detected among those compounds tested with both QB RNA and 4X174 DNA as the infectious agents. The results in Table 2 suggest the following: when the 7-methyl group of Me2BA is brominated or substituted by an oxiranyl group, the inhibitory phenomenon is strongly enhanced, the latter modification being more effective for inhibition than the former. The replacement of one or both methyl groups of Me2BA with formyl groups, or that Af the 7-methyl group with a hydroxymethyl group, results in in appreciable reduction of the potency of Me2BA. Whereas :he presence of quinone or hydroxyl groups at the 5,6-position K-region) of Me2BA lowers the inhibitory response, the introduction of a 5,6-oxide function markedly stimulates inhi-
Proc. Natl. Acad. Sci. USA 74 (1977)
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Table 1. Effect of PAH and their derivatives on the infectivity of QB RNA (method A) Concentration
Compound None BA
Me2 BA Me2 BA-5,6-oxide
Infec% Inhibition
(g/ml)
tivity (PFU)
0 0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0
310 327 454 247 285 129 5 347 296 70
0 0 0 20 8 58 99 0 6 77
0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0
272 306 230 306 263 91 352 274 59
12 1 26 1 15 71 0 12 81
0.1 1.0 10.0
171 2 0
45 99 >99
0.1 1.0 10.0
183 56 0
41 82 >99
Phenanthrene-
9,10-oxide BP
BP-4,5-oxide anti-BP-diolepoxide
syn-BP-diolepoxide
QB RNA (0.01 ,g) and a given PAH, in the amounts shown above, combined in a total volume of 0.1 ml containing 2% dimethylformamide. To this mixture was added an equal volume of E. coli spheroplasts (method A). After 5 min at 250, this mixture was plated on agar with E. coli Q13 as indicator and placed in an incubator at 370 overnight. The plaque values (PFU = plaque-forming units) shown represent an average for two plates. The concentration (,ug/ml) represents the amount of PAH present in 1 ml of the final infection were
mixture.
bition. The opening of the K-region of Me2BA results in the loss of its biological activity. Simultaneous trans-hydroxylation at C-8,9 and epoxidation at the C-9,10 in the anti-configuration resulted in a pronounced increase in the activity of BA, whereas a similar oxidative transformation to the syn-isomer had no effect on the inhibitory response. Table 2 also indicates that introduction of hydroxyl groups at the 4, 7, or 8 position of BP has little or no effect on the biological activity of kX174 DNA. Whereas the addition of quinone or of hydroxyl groups to the 4,5 positions fails to show any significant influence on biological response, the presence of an epoxide group across these positions results in a marked increase in the activity of BP. Trans-hydroxylation at the 7,8 positions of BP produces further enhancement of activity. Introduction of an epoxide function to various positions of BP had variable effects. Thus, whereas an epoxide group at the K-region or the 9,10 positions resulted in little or no change in activity, the 7,8-epoxy derivative was strongly inhibitory. As was observed with QB RNA, the 7,8-diol-9,10-epoxide derivatives of BP are also highly inhibitory, the anti-isomer being the most active compound so far tested. Phenanthrene and its three oxide derivatives were noninhibitory. Fig. 1 illustrates the dose response of OXX174 DNA to the two diolepoxide isomers of BP and BA when assayed by modified
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Proc. Natl. Acad. Sci. USA 74 (1977)
Table 2. Effect of PAH derivatives on infectivity of OX174 DNA (modified method A) % Inhibition
Compound Phenanthrene Phenanthrene-9,10-oxide 1-Methylphenanthrene9,10-oxide Benzo [c I phenanthrene9,10-oxide BA Me2BA 7-Bromomethyl-12-MeBA
7-Hydroxymethyl-12-MeBA 7,12-Diformyl-BA 7-Formyl-12-MeBA 12-Formyl-7-MeBA Me2 BA-5,6-dione Me2 BA-5,6-diol 7-Oxiranyl-12-MeBA 1,4-Dimethyl-2-phenylnaphthalene-3,2' -dicarboxaldehyde 7-Hydroxymethyl-12MeBA-5,6-oxide BA-5,6-oxide anti-BA-diolepoxide
syn-BA-diolepoxide BP 4-HO-BP
7-HO-BP 8-HO-BP BP-4,5-dione BP-4,5-dlol
BP-7,8-diol BP-4,5-oxide 7,8-Epoxy-H4BP
9,10-Epoxy-H4BP anti-BP-diolepoxide syn-BP-diolepoxide
at 0.2 mg/ml
0 0
100
at 2.0 ,4g/ml
7
anti - BA- diolepoxideo
0 0 0 3 28 72 82 29 20 35 52 44 8 >99
10
24
12 43 44 0 14 12 28 0 0 0 40 24 48 23 95 35
70 >99 97 12 54 9 41 20 0 7 92 61 >99 35 >99 77
The assay was similar to that described in the footnote for Table 1, except that 4X174am3 DNA was used as the test nucleic acid and E. coli spheroplasts were preinfected with this DNA. Two-tenths milliliter of the infected spheroplasts was introduced into tubes containing PAH in 4 jil of dimethylformamide; after 5 min at 250, the spheroplasts were plated with E. coli HF4714 as the indicator (modified method A). For the preinfection, equal volumes of E. coli spheroplasts and OX174 DNA were mixed in 5 mM EDTA and kept at 40 until used. The concentration of the ihfecting DNA was approximately 0.03 jg/ml, and 0.2 ml of infected spheroplasts yielded between 100 and 300 plaques in the absence of PAH after plating. Percent inhibition was calculated from the average number of plaques observed from triplicate plates.
method A. The concentrations of BP-diolepoxide required for 50% inactivation of infectivity (150) are approximately 0.04 ,ug/ml (0.13 ,AM) and 0.6 Ag/ml (2 ,uM) for the anti- and synforms, respectively. The I'50 molar concentrations are probably lower than the values derived from this experiment, since stability studies with each isomer in aqueous media (data not shown) indicate that both isomers are unstable, the syn-form being less stable than the anti-form. The I50 for the anti-form of the BA-diolepoxide derivative was approximately 2 ,uM, whereas the syn-BA-diolepoxide demonstrated no significant inhibition. The stability of the BA-derivatives in aqueous solution was not examined.
syn-BP-diolepoxide
o C
5 19 36 0 0 4 13 10 0 52
0
.~50
C
syn- BA - diolepoxide
0
0.5
1.0
1.5
2.0
ing of PA H per m u FIG. 1. Effect of the diolepoxides of BP and BA on the infectivity of OX174 DNA. The assay for infectivity was done by modified method A. The concentrations of the anti- and syn-forms of 7,8dihydroxy-9,10-epoxy-BP and 8,9-dihydroxy-10,1t-epoxy-BA in the E. coli spheroplast mixture (0.2 ml) were as indicated. The diolepoxides were the (+) racemic mixtures. Three nanograms of DNA from the amber phageoX174am3 were used as infectious nucleic acid in the same reaction mixture. Each point represents values calculated from the average of three plates.
Using the same assay procedure (modified method A), Fig. 2 shows that the separated (-) and (+) stereoisomers of antiBP-diolepoxide exhibit essentially the same inhibitory dose response as the racemic mixture. In order to distinguish between active aromatics that are
capable of reacting directly with nucleic acid molecules and those that are inactive but can be activated by intracellular modification, we devised a nucleic acid "pretreatment" procedure (method B). In this procedure, the infectious nucleic acid
1oo0 4.. C
C
al
.' 50
0.5 szg of anti - BP - diolepoxide per ml
FIG. 2. Effect of the (-) and (+) isomers of anti-BP-diolepoxide on the infectivity of OX174 DNA. The assay procedure and conditions of infection were the same as in the legend of Fig. 1 except that the separated (-) and (+) isomers of anti-7,8-dihydroxy-9,10-epoxy-BP were used, at the concentrations shown above, in reaction mixtures with OX174am3 DNA.
Biochemistry:
Proc. Natl. Acad. Sci. USA 74 (1977)
Hsu et al.
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Table 3. Effect of OX174 DNA pretreatment with PAH derivatives on infectivity Compound
None BA Me2BA 7-Bromomethyl-12-MeBA 12-Formyl-7-MeBA 7-Oxiranyl-12-MeBA 7-Hydroxymethyl-12-MeBA5,6-oxide Me2 BA-5,6-oxide anti-BA-diolepoxide
syn-BA-diolepoxide BP BP-7,8-diol
7,8-Epoxy-H, BP 9,10-Epoxy-H4BP anti-BP-diolepoxide syn-BP-diolepoxide
Phage titer (PFU)
% Inhibition
108 100 43 110 41
0 0 7 60 0 62
115 20 24 100 99 80 107 111 0 60
0 81 78 7 8 26 0 0 >99 46
105
0
0.05
0.10
0.15
0.20
pg of anti-BP-diolepoxide
,X174am3 DNA (10 Mg) and 0.5 ig of PAH were mixed in a final volume of 0.1 ml and allowed to stand at 370 for 10 min. The DNA was reisolated after treatment with solvents as described under method B in Methods, and assayed for infectivity. Recovery of DNA after solvent treatment was nearly quantitative.
FIG. 3. Effect on infectivity of QB RNA pretreated with different concentrations of anti-BP-diolepoxide. QB RNA (10 Mg) was mixed with varying concentrations of anti-BP-diolepoxide in a final volume of 0.05 ml containing 0.01 M Tris-HCl (pH 7.5) and 4% dimethylformamide. After 10 min at 370, the unreacted hydrocarbon was removed by ethanol precipitation and acetone washing (method B). QB RNA was dissolved in 1 ml of water, its concentration was determined, and, after appropriate dilution, it was assayed in triplicate for infectivity with spheroplasts.
is preincubated with PAH and unreacted hydrocarbon is removed by solvent treatment prior to spheroplast exposure. Table 3 shows that when 4X174 DNA was pretreated with BA, Me2BA, 12-formyl-7-MeBA, 7-hydroxymethyl-12-MeBA5,6-oxide, syn-BA-diolepoxide, BP, BP-7,8-diol, 7,8-epoxyH4BP, and 9,10-epoxy-H4BP and then assayed after removal of the unreacted compounds, little, if any loss of infectivity occurred. These results suggest that none of the above compounds is capable of inactivating infectious nucleic acids unless it is converted to some chemically reactive form after incorporation into spheroplasts, as was demonstrated in the results of Table 2. Exceptions are BA, syn.BA-diolepoxide and 9,10epoxy-H4BP which are relatively inactive even when assayed by the modified method A procedure (Table 2). The remaining compounds listed in Table 3 are effective inhibitors of OXX174 DNA expression when they are assayed by the pretreatment procedure; hence, these compounds are capable of interacting with and modifying nucleic acid molecules directly without intracellular activation. Similar results, with the same compounds, were found when QB RNA served as the infectious agent. It is important to note that, in control experiments, when E. coli tRNA was pretreated with anti-BP-diolepoxide and extracted by the solvent treatment procedure used in the above assay, no residual inhibitory activity could be detected in the recovered RNA fraction upon the addition of test infectious nucleic acid. Fig. 3 shows the effect of increasing concentration of antiBP-diolepoxide on the expression of QB RNA as determined by the pretreatment procedure. For 10 ,gg of the viral RNA, almost complete loss of infectivity (97%) was achieved by exposure to 0.1 jig of the diolepoxide, and 50% inhibition occurred at a molar ratio of RNA nucleotide to BP-diolepoxide of approximately 300:1. DISCUSSION Recently, it has been shown that certain derivatives of PAH can react covalently with nucleic acids, in vivo and in vitro, providing a possible explanation for the way in which these chemical agents elicit biological responses in animal and mi-
crobial organisms (16-18). In this report, we have reexamined the application of a phage assay system, previously described by this laboratory (1), for the detection of biologically active aromatics. The results described here are consistent with our earlier findings and indicate that certain compounds, known to be carcinogenic in animals, are also capable of blocking the replication of infectious nucleic acids in E. coli spheroplasts. The phage nucleic acid may be either RNA or DNA, and the assay system is suitable for quantitating the relationship between dose and biological response for various active hydrocarbons. Repeat experiments indicate that, although the plaque titer assay is highly reproducible, differences of 20% are not significant. Pretreatment of the infectious nucleic acid with the hydrocarbon derivatives and then removal of unreacted compound prior to assay allows one to discriminate between those aromatics that can inactivate infectious nucleic acids directly and those that require metabolic activation to demonstrate an inhibitory response. Presumably, direct inactivation of infectivity occurs with aromatics that are capable of binding nucleic acid molecules without any chemical alteration, whereas aromatics that require exposure to spheroplasts become reactive only after intracellular modification. In general, those aromatics that exhibit the highest activity in the assay procedures described here are those known to function as alkylating agents capable of direct covalent interaction with nucleic acids. These include the K-region oxides and the non-K-region diolepoxides, both of which have been shown to be metabolites of the parent hydrocarbon formed by microsomal enzymes (17-21). Neither 7,8-epoxy-H4BP nor 9,10-epoxy-H4BP is apparently capable of modifying 4X174 DNA directly. The former derivative, however, can be converted into an active form by E. coli spheroplasts. These results either indicate the importance of the position of the epoxide group or reflect the instability of the 9,10-epoxy-H4BP. The other particularly active compounds are the 7-bromomethyl and 7-oxiranyl derivatives of 12-MeBA; these are not metabolic products, but are expected, on chemical grounds, to be effective
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alkylating agents. The former derivative has been shown to react with nucleic acids (22), and both compounds are known to be carcinogenic (22, 23). Recently, in the process of BP-induced carcinogenesis, BP-diolepoxide has been implicated as the ultimate carcinogen since it appears as a conjugate of nucleic acids when BP is incubated with cells in culture (19-21, 24-27). Osborne et al. (26) found that both anti- and syn-BP-diolepoxide isomers react readily with DNA, yielding similar products. Although preliminary data indicate its carcinogenicity to be weak (S. Hecht, unpublished data), presumably due to its instability, the antiBP-diolepoxide is the most active mutagenic derivative synthesized thus far (19, 28). Our experiments are in general agreement with the above observations. The two stereoisomers of BP-7,8-diol-9,10-epoxide react directly with QB RNA and (DX174 DNA to inactivate both nucleic acids; the anti-form is the most effective inhibitor of infectivity examined in this study. Under the conditions used for the pretreatment procedure, approximately 14 molecules of diolepoxide are required for each molecule of QB RNA if 50% inactivation is to be achieved (Fig. 3). The studies with the two isomers of BA-diolepoxide show that the anti-form has a strong inhibitory effect on viral replication, whereas the synform has little or no effect. These results suggest that the diolepoxide derivatives of BP and BA are highly active as inhibitors of the replication of infectious nucleic acid; the geometric configuration of these isomers appears to be an important determinant for the degree of biological activity expressed. The (-) and (+) isomers of anti-BP-diolepoxide showed little difference in their inhibitory response. It is possible that some of the results obtained with these highly reactive compounds also reflect relative differences in their stability. Recently, microorganisms and cultured animal cells have been adopted, with increasing frequency, for the detection and identification of toxic chemical substances (29). The use of biologically active nucleic acids, in the manner described here, may serve a similar purpose. Because of the rapidity and economy of evaluating the hydrocarbon response, the phage assay system is well suited for the monitoring of large numbers of compounds. It also has an advantage for detecting highly reactive, but unstable, derivatives of PAH, which might go undetected in other test systems yet be important intermediates in the expression of PAH toxicity. In addition to certain obvious differences between the assay system reported here and the Salmonella typhimurium-mutagen system (30), there appears to be a difference in the response to specific hydrocarbons. For example, BP and Me2BA are not mutagenic in the Salmonella reversion test unless there is prior activation by mammalian enzymes (31); however, these same compounds are inhibitory for phage replication in E. coli spheroplasts. Schumm (3) and Fried (4) identified various oxidation products of labeled Me2BA after its incubation with E. coli spheroplasts. The disparity between E. coli and S. typhimurium in their response to certain hydrocarbons may reflect metabolic differences for these two organisms, or may result from the altered permeability properties of lysozyme-formed E. coli spheroplasts used in this study. Nonetheless, the preincubation of hydrocarbons with mammalian extracts, as well as the use of certain bacterial mutants deficient in DNA excision repair (32), may improve the sensitivity and broaden the scope for detecting potentially harmful substances in the infectious nucleic acid-spheroplast system. The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the United States Energy Research and
Proc. Natl. Acad. Sci. USA 74 (1977) Development Administration under Contract no. EY-76-C-02-0069. This study was partly supported by a grant from the American Cancer Society (BC-132A) and by funds from EPA Subagreement no. 77BAN. We thank Drs. H. Cho and K. B. Sukumaran and Ms. Cecilia Cortez for furnishing samples of some of the compounds used. 1. Hsu, W. T., Moohr, J. W. & Weiss, S. B. (1965) Proc. Natl. Acad. Sci. USA 53,517-524. 2. Hsu, W. T., Moohr, J. W., Tsai, A. Y. M. & Weiss, S. B. (1966) Proc. Natl. Acad. Sci. USA 55, 1475-1482. 3. Schumm, D. L. (1969) Ph.D. Dissertation, University of Chicago. 4. Fried, J. (1974) in Chemical Carcinogenesis, eds. T'so, P. 0. P. & di Paolo, J. A. (Dekker, New York), pp. 197-215. 5. Eoyang, L. & August, T. P. (1968) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. 12, Part B, pp. 530-540. 6. Goh, S. H. & Harvey, R. G. (1973) J. Am. Chem. Soc. 95,242243. 7. Harvey, R. G., Goh, S. H. & Cortez, C. (1975) J. Am. Chem. Soc. 97,3468-3479. 8. Cho, H. & Harvey, R. G. (1974) Tetrahedron Lett., 14911494. 9. Beland, F. A. & Harvey, R. G. (1976) J. Chem. Soc. Chem. Commun., 84-85. 10. McCaustland, D. J. & Engel, J. F. (1975) Tetrahedron Lett., 2549-2552. 11. Harvey, R. G., Pataki, J., Wilke, R. N., Flesher, J. W. & Soedigdo, S. (1976) Cancer Lett. 1, 339-344. 12. Sims, P. (1973) Biochem. J. 131, 405-413. 13. Hadler, H. I. & Kryger, A. C. (1960) J. Org. Chem. 25, 18961901. 14. Pataki, J., Wlos, R. & Cho, Y. (1968) J. Medic. Chem. 11, 1083-1086. 15. Boyland, E. & Sims, P. (1965) Biochem. J. 95,780-787. 16. Miller, E. C. & Miller, J. A. (1974) in The Molecular Biology of Cancer, ed. Busch, H. (Academic Press, New York), pp. 377402. 17. Sims, P. & Grover, P. L. (1974) Adv. Cancer Res. 20, 165-274. 18. Heidelberger, C. (1975) Annu. Rev. Biochem. 44,79-121. 19. Huberman, E., Sachs, L., Yang, S. K. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73, 607-611. 20. King, H. W. S., Osborne, M. R., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Proc. Natl. Acad. Sci. USA 73,2679-2681. 21. Yang, S. K., McCourt, D. W., Roller, P. P. & Gelboin, H. V. (1976) Proc. Natl. Acad. Sci. USA 73,2594-2598. 22. Dipple, A., Brookes, P., MacKintosh, D. S. & Rayman, M. P. (1971) Biochemistry 10,4323-4330. 23. Flesher, J. W., Harvey, R. G. & Sydnor, K. (1976) Int. J. Cancer 18,351-353. 24. Daudel, P., Deuquesne, M., Vigny, P., Grover, P. L. & Sims, P. (1975) FEBS Lett. 57, 250-253. 25. Ivanovic, V., Geacintov, N. E. & Weinstein, I. B. (1976) Biochem. Biophys. Res. Commun. 70, 1172-1179. 26. Osborne, M. R., Thompson, M. H., Tarmy, E. M., Beland, F. A., Harvey, R. G. & Brookes, P. (1976) Chem. Biol. Interact. 14, 343-348. 27. Weinstein, I. B, Jefferey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H. & Nakanishi, K. (1976) Science 193, 592-595. 28. Newbold, R. F. & Brookes, P. (1976) Nature 261,52-54. 29. Montesano, R., Bartsh, H. & Tomatis, L., eds. (1976) Screening Test in Chemical Carcinogenesis (International Agency for Research on Cancer, World Health Organization, Lyon), IARC Scientific Publication no. 12. 30. McCann, J., Choi, E., Yamasaki, E. & Ames, B. N. (1975) Proc. Natl. Acad. Sci. USA 72,5135-5139. 31. Ames, B. N., Durston, W. E., Yamasaki, E. & Lee, F. D. (1973) Proc. Natl. Acad. Sci. USA 70,2281-2285. 32. Ames, B. N., Lee, F. D. & Durston, W. E. (1973) Proc. Natl. Acad. Sci. USA 70, 782-786.
