We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.

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Covalent binding of the benzo[alpyrene metabolite (+) 7#,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[alpyrene diol epoxide) and exocyclic amino groups of the respective purines.The potent carcinogen benzo[a]pyrene (BzaP) undergoes metabolism in vivo to a chemically reactive intermediate, which can then react with cellular RNA, DNA, and protein (1, 2). It is widely accepted that these covalent interactions with cellular macromolecules, particularly DNA, are an essential initial step in the process of carcinogenesis (3,4). Recent work has implicated 7/3,8a-dihydroxy-9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene (BzaP diol epoxide, Structure I) as the 12 I molecular species responsible for binding to nucleic acids in vduo (5-7). Weinstein and coworkers have shown that the major RNA adduct found in cell culture is identical to the product obtained by reacting BzaP diol epoxide with poly(G), and involves a covalent bond between C-10 of the hydrocarbon and the N2 exocyclic amino group of guanine (8, 9). A similar structure has been reported for reaction of the isomeric diol epoxide, 703,8a-dihydroxy-90,10,B-epoxy-7,8,9, 10-tetrahydrobenzo[a]-pyrene, with poly(G) (10). The structures of the products obtained in the reaction between BzaP diol epoxide and DNA have not been reported, primarily because only microgram quantities of such adducts can be readily isolated from the analogous reaction with DNA.We have isolated seven distinct products from the reaction of BzaP diol epoxide with calf thymus DNA, and by the use of high-sensitivity, high-resolution mass spectrometry the structures of these products were determined. The results show that adducts are formed between BzaP diol epoxide and the bases guanine, adenine, and cytosine, and involve reactions with the exocyclic amino group of guanine and adenine.MATERIALS AND METHODS Adduct Formation and Isolation. Formation of microsomal enzyme-activated BzaP-DNA adducts has been described (11). Racemic BzaP diol epoxide was synthesized as described (12).Diol epoxide (12 nmol/mg of DNA) in 100 Al of dimethylsulfoxide was added to a solution of 100 mg of calf thymus DNA (Sigma Chemical Co.) in 100 ml of 10 mM phosphate buffer (pH 7.2) at 37'. After 24 hr, unbound BzaP diol epoxide and its hydrolysis products were removed by ethyl acetate extraction. The DNA was then precipitated with ethanol and the precipitate was heated to remove intercalated material. The DNA was enzymatically hydrolyzed with deoxyribonuclease II, spleen phosphodiesterase, and alkaline phosphatase (Sigma Chemical ...

A monooxygenase isolated from 5-day old etiolated Vinca rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was inpendent upon NADPH (neither NADH nor combination of NADH, NADP+ and ATP served as substitutes) and O2. Geraniol hydroxylation was enhanced by dithiothreitol (monothiols were less effective) and inhibited by phospholipases, thiol reagents, metyrapone, and cytochrome c, as well as other inhibitors of cytochrome P-450 systems. Geraniol was hydroxylated at a faster rate than nerol, but the alcohols possessed similar apparent Km values. The membrane-bound hydroxylase was solubilized by treatment with sodium cholate, Renex-30, or Lubrol-WX. Cholate-treated enzyme was resolved by DEAE-cellulose chromatography and reconstitution of the hydroxylase was effected utilizing different fractions containing cytochrome P-450, a NADPH-cytochrome c reductase, and lipid.

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