A previous report from this laboratory has shown that certain derivatives of polycyclic aromatic hydrocarbons bind to 4X174 DNA and render it noninfectious. The present work describes the relationship between the extent of 4X174 DNA binding by (±) -anti-benzo [alpyrene-7,8-dihydrodiol-9,10- (1-3), it reacts covalently with nucleic acids (4, 5), and DNA conjugated with anti-BPdiolepoxide has been isolated from cultured cells exposed to BP (2, 6). In addition, several studies have shown that BP-diolepoxide is unusually active as a mutagenic agent for mammalian (1, 7) and bacterial cells (8,9), and a more potent carcinogen than the parent hydrocarbon in newborn mice (10).We have described a bacteriophage assay system for evaluation of the biological activity of polycyclic aromatic hydrocarbons (PAH) and related compounds (11)(12)(13) (14), except that the infected cell lysates obtained by lysozyme treatment were subjected to two successive centrifugations (15 min at 17,000 X g and 2 hr at 100,00 X g) for sedimentation of phage. The resuspended phages were then purified as reported (14). DNA was isolated from purified viruses by several extractions with phenol containing 1% sodium dodecyl sulfate, followed by dialysis against 10 mM Tris (pH 7.5)/1 mM EDTA. The concentration of OX DNA was determined on the basis that, in a 1-cm light path, 1 A260 unit is equivalent to 36 jsg/ml or 1.3 X 1013 molecules per ml (14).Radioactive 4X174am3 DNA was prepared by the same procedure described above, except that infection was carried out in the presence of [3H]thymidine (6 Ci/mmol) at a concentration of 20 nmol/ml. Isolation of DNA from the radioactive virus by phenol extraction gave a specific activity of 3 X 104 cpm/,ug of DNA.Infectivity Assay of gbX174am3 DNA. The infectivity of OX174am3 DNA was assayed by absorption with spheroplasts prepared from E. coli K12W1485 and plating with its sensitive host, E. coli HF4714, for plaque development, as reported (11, 13). Preparation of Radioactive Replicative Intermediates RF1 and RF2. 3H-Labeled 4X174am.3 phage, prepared as described above, was used to infect E. coli C in the presence of chloramphenicol (15 ug/ml). After 10 min of infection, DNA was extracted from the infected cells by the procedure of Brutlag et al. (15). The cell lysate was adjusted to contain 1 M NaCl and was extracted once with phenol; the nucleic acid was precipitated from the aqueous phase with ethanol. After resuspension in Tris/EDTA and sedimentation in a neutral sucrose gradient, the pellet showed two radioactive peaks, 21 S and 16 S, corresponding to the qX174 DNA replicative intermediates RF1 and RF2, respectively.

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 replication.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) 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, trans-4,5-dihydroxy-4,5-dihydro-BP; 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...

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