A bacteriophage system for screening and study of biologically active polycyclic aromatic hydrocarbons and related compounds.

Abstract

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...

Modification of deoxyribonucleic acid by a diol epoxide of benzo[a]pyrene. Relation to deoxyribonucleic acid structure and conformation and effects on transfectional activity

“…Single-hit kinetics were also found in the study with 0X174 phage DNA (Hsu et al, 1977a). In vitro studies also provide evidence that BPDE I modified plasmid DNA and BPDE I modified 0X174 DNA are impaired in their template activity for DNA replication (Mizusawa & Kakefuda, 1979;Hsu & Berg, 1978). Because of its simplicity, the plasmid DNA transfection assay described in the present study is well suited for the detection and identification of other toxic chemical substances that bind to DNA.…”

“…Our data on the inactivation of transfection by BPDE I modification of plasmid DNA are consistent with single-hit kinetics. Single-hit kinetics were also found in the study with 0X174 phage DNA (Hsu et al, 1977a). In vitro studies also provide evidence that BPDE I modified plasmid DNA and BPDE I modified 0X174 DNA are impaired in their template activity for DNA replication (Mizusawa & Kakefuda, 1979;Hsu & Berg, 1978).…”

“…values reported by Hsu & Berg (1978). The distributions of BPDE I residues in unrestricted DNA and in the separated fragments are expressed as the number of BPDE I residues bound per 104 bases.…”