Metabolites of polycyclic aromatic hydrocarbons. II. Synthesis of 7,8-dihydrobenzo[]pyrene-7,8-diol and 7,8-dihydrobenzo[]pyrene-7,8-epoxide

Three diastereomeric pairs of diol epoxides, two tetrahydro-epoxides, and the K-region oxide of the polycyclic aromatic hydrocarbon benz[alanthracene were evaluated for mutagenic activity in strain TA 100 of Salmonella typhimurium and in line V79-6 of Chinese hamster lung cells. The two diastereomeric 1,2-epoxides of the trans-3,4-dihydrodiol of benz[alanthracene are 15 to 35 times more mutagenic to the bacteria and 65 to 125 times more mutagenic to the mammalian cells than are the diastereomeric pairs of benz[ajanthracene-8,9-diol-10,11-epoxides or benz[alanthracene-10,11-diol-8,9-epoxides. 1,2-Epoxy-1,2,3,4-tetrahydrobenzlajanthracene is the most mutagenic and cytotoxic of the nine derivatives and is 5 and 25 times more mutagenic than 3,4-epoxy-1,2,3,4-te-

contrasting

confidence: 65%

Mutagenicity and cytotoxicity of benz[ a ]anthracene diol epoxides and tetrahydro-epoxides: Exceptional activity of the bay region 1,2-epoxides

Wood

Chang

Levin

et al. 1977

“…Crystalline BaP diol epoxide was synthesized essentially by the method of McCaustland and Engel (11)…”

Section: Methodsmentioning

confidence: 99%

DNA alkylation and unwinding induced by benzo[a]pyrene diol epoxide: modulation by ionic strength and superhelicity.

Gamper¹,

Straub²,

Calvin³

et al. 1980

Superhelical and partially relaxed DNAs of simian virus 40 were allowed to react in vitro with (1)7#,8a-dihydroxy-9a, 8,9, [3H]BaP diol epoxide, 1.5 mg/ml in 19:1 (vol/vol) tetrahydrofuran/triethylamine, was also synthesized according to published procedures (12). It had a specific activity of 1.23 Ci/mmol (1 Ci = 3.7 X 1010 becquerels). The BaP diol epoxide stocks were stored at -70'C and dilutions were made with dimethyl sulfoxide. SV40 DNA. Superhelical (form I) SV40 DNA was isolated from infected TC-7 African green monkey cells by a modification of the Hirt extraction essentially as described by Hallick et al. DNA Alkylation and Adduct Characterization. SV40 DNA was modified with BaP diol epoxide in 20 mM Tris-HCl (pH 8.0)/0.5 mM EDTA/10% (vol/vol) dimethyl sulfoxide at the indicated molar reaction ratios (BaP diol epoxide to DNA mononucleotide). Reaction mixtures were incubated in the dark at 370C for at least 90 min. Alkylation was quantified by radioactivity counting, using SV40 [14C]DNA and [3H]BaP diol epoxide. The modified DNA was diluted to 1.0 ml with 20 mM Tris-HCl (pH 8.0)/0.5 mM EDTA/0.5 M NaCI and extracted three times with 1.0 ml of ethyl acetate. After addition of 30 ,ug of carrier calf thymus DNA (Sigma), the DNA was precipitated from the aqueous phase by addition of 2.0 ml of NaClsaturated ethanol and by storage overnight at -20°C. The DNA was pelleted, washed, and resuspended in 1.0 ml of NaCl-saturated 70% (vol/vol) ethanol. It was then collected on a Millipore 0.45-,um-pore-diameter HAWP filter, combusted, and assayed for radioactivity. Binding data were calculated by assuming SV40 DNA contains 5200 base pairs (14) and has a molecular weight of 3.6 X 106(15).Hydrocarbon adducts, enzymatically released from alkylated DNA, were isolated by Sephadex LH-20 chromatography and further resolved by high-pressure liquid chromatography as described by Straub et al. (8).Gel Electrophoresis. Vertical 1.4% agarose (Bio-Rad) slab gels (13.3 X 13.0 X 0.5 cm) were prepared and run in 40 mM Tris-HCl (pH 7.9)/5 mM sodium acetate/i mM EDTA at 50 V for [12][13][14][15][16] hr. Composite 2% polyacrylamide/0.5% agarose Abbreviations: BaP diol epoxide, 8,9, SV40, simian virus 40.

“…In this procedure, referred to as the "pretreatment" method, infectious nucleic acid and hydrocarbon were mixed together and incubated at 370 for 10 (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).…”

Section: Methods Preparation Of Spheroplasts and Infectious Nucleic Amentioning

confidence: 99%

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

Hsu¹,

Harvey²,

Lin³

et al. 1977

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...