Polyguanylic Acid Research Articles

Nucleic acid base grafted polyethylenimine. Cytosyl pendant groups: Elucidation of structure with solution spectroscopy

AbstractIn a previous paper, potassium 2‐(cytos‐1‐yl)propanoate was grafted onto polyethylenimine of various molecular weights. These water‐soluble polynucleotide analogs exhibited remarkable properties in their solution ultraviolet spectra. High hypochromicity values of 50% and 54% have been observed in alkaline aqueous solution and 50% trifluoroethanol solution, respectively. Percent hypochromicity value was observed to be directly proportional to percent graft. Red shift and hyperchromicity as a function of pH were observed for both the monomer model and the graft polymers. Light‐scattering experiments on nucleic acid base grafted hydrophilic polymer backbones were carried out. Radius of gyration and second virial coefficient values indicated stiff macromolecules, rodlike in nature. Therapeutic potential of cytosyl grafted polyethylenimine was suggested by the results from continuous mixing experiments with polyinosinic acid and polyguanylic acid. Job plots showed enhanced hypochromicity, whose magnitude was dependent upon the polynucleotide and the base‐base distance.

Nucleic acid base grafted poly(ethylenimine): Polynucleotide analog and therapeutic agent

Poly(N-acyl ethylenimine) of various molecular weights was synthesized by the cationic ring-opening polymerization of 2-H-2-oxazoline and 2-methyl-2-oxazoline. Acid hydrolysis afforded the linear poly(ethylenimine) which was used in the following grafting reactions. Vapor pressure osmometry, gel permeation chromatography and viscosity measurements were used to characterize the polymers synthesized. Potassium 2-(cytos-l-yl) propanoate and potassium 3-(cytos-l-yl) butanoate were synthesized in good yield from thenucleic acid base. These cytosyl pendant groups were grafted onto the poly(ethylenimine) using 4-chloro-1-(4-chlorobenzenesulfonyl) benzotriazole, 90% graft, and norborn-5-ene-2,3-carboximido diphenyl phosphate, 70% graft. Grafting of the t-butoxycarbonyl and n-butoxycarbonyl protected cytosyl pendant groups resulted in a 47% graft. Hypochromicity studies indicated an ordered structure at the local level. The secondary structure is a base-stacked conformation. Light scattering results gave insight into the tertiary structure of the macromolecule. The polymer exists in solution as stiff chains, rod-like in nature. Continuous mixing experiments were carried out with polyribonucleotides. The Job plots indicated increased level of ordered conformation with either an increase in the molecular weight or an increase of the base to base distance in the analog. Enhancement in the base-stacked conformation was higher in the polyinosinic acid complex than that of the polyguanylic acid complex.

Glutathione plus cytosol- and microsome-mediated binding of 1,2-dichloroethane to polynucleotides

1,2-[1,2- 14C]Dichloroethane was metabolized by rat hepatic microsomes to products that irreversibly bound polynucleotides. The polynucleotides were then enzymatically hydrolyzed and the products separated by a high-performance liquid chromatograph (HPLC) equipped with an ODS or a SCX column. The products of microsome-mediated binding were identified in the HPLC eluate as 1, N 6 -ethenoadenosine to polyadenylic acid, 3, N 4 -ethenocytidine to polycytidylic acid, and two cyclic derivatives to polyguanylic acid. 1,2-[1,2- 14C]Dichloroethane was also metabolized in the presence of a glutathione (GSH)-cytosolic fraction and a polynucleotide. After enzymatic hydrolysis of the polynucleotide, the major peak of radioactivity was eluted from a Sephadex G-25 column in the salt volume which would exclude the presence of a product containing both GSH and a nucleoside. Chromatography by ODS-HPLC of the major peak from Sephadex G-25 indicated the presence of a GSH metabolite of 1,2-dichloroethane that did not contain a nucleoside. A similar hydrophilic peak was obtained for the hydrolysis products of polynucleotides from a glutathione plus cytosol incubation in which the polynucleotide instead of being added prior to the incubation was added after the incubation. The products of the glutathione plus cytosol metabolism of 1,2-[1,2- 14C]dichloroethane appear to be glutathione metabolites that coisolated with the polynucleotides rather than covalently bound adducts. In conclusion, covalently bound adducts were identified for microsomemediated binding of 1,2-dichloroethane to polynucleotides, while no evidence was obtained for glutathione plus cytosol-mediated covalent binding to polynucleotides.

Metabolic activation of mutagenic N-hydroxyarylamines by O-acetyltransferase in Salmonella typhimurium TA98

A new enzymatic activation of mutagenic N-hydroxyarylamines is described. An acetyl-CoA dependent enzyme that can activate 2-hydroxyamino-6-methyldipyrido[1,2- a: 3′,2′- d]imidazole ( N-OH-Glu-P-1) to a reactive species capable of binding to nucleic acid was found in a cell-free extract of Salmonella typhimurium TA98 but not in that of TA98/1,8-DNP 6, which shows low sensitivity to the mutagenic activity of N-OH-Glu-P-1. The enzyme was partially purified by streptomycin treatment, ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration chromatography of Sephadex G-150. Its molecular weight was estimated to be approximately 48,000. The directly mutagenic N-hydroxyarylamines, such as N-OH-Glu-P-1, 3-hydroxyamino-1-methyl-5 H-pyrido[4,3- b] indole ( N-OH-Trp-P-2), and N-hydroxy-2-aminofluorene ( N-OH-AF), were transformed to reactive derivatives by partially purified enzyme in the presence of acetyl-CoA. The K m value for acetyl-CoA was calculated to be 3.3 μ m. No acetyl residue, however, was incorporated into nucleic acid adducts. The enzymatic product of N-OH-Glu-P-1 bound most efficiently to polyguanylic acid among four polynucleotides. The enzyme did not show the N,O-acetyltransfer activity of N-hydroxyacetylaminofluorene ( N-OH-AAF). These results indicate that the enzymatic product of N-hydroxyarylamine is N-acetoxyarylamine, and that this enzyme can be called acetyl-CoA: N-hydroxyarylamine O-acetyltransferase. O-Acetyltransferase activity was inhibited by SH-blocking agents, several phenolic compounds, such as pentachlorophenol and 1-nitro-2-naphthol, and an antibiotic thiolactomycin. S. typhimurium mutation studies suggested that the O-acetyltransferase functions as an enzyme activating certain N-hydroxyarylamines within bacterial cells and is involved in the formation of mutants.

Interaction of nucleophiles with the enzymatically-activated carcinogen, N-hydroxy-2-acetylaminofluorene, and with the model ester, N-acetoxy-2-acetylaminofluorene.

A new method was devised to study adduct formation of N-acetyl-L-methionine (N-Ac-met) with activated species of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF). Following degradation of the adducts, the isomeric methylthio derivatives of 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) were separated and quantified by h.p.l.c. With the use of this method the nucleophilicity of N-Ac-met toward enzymatically produced N-acetoxy and N-sulfonoxy derivatives of 2-AF and 2-AAF, respectively, and toward the synthetic model ester, N-acetoxy-2-acetylaminofluorene (N-OAc-2-AAF), was determined. For comparison, the interaction of tRNA and polyguanylic acid (poly G) with the above compounds was measured by standard procedures. tRNA was an efficient acceptor of enzymatically formed N-acetoxy and N-sulfonoxy derivatives as well as of N-OAc-2-AAF. The nucleophilicity of N-Ac-met toward enzymatically formed N-sulfonoxy-2-AAF was comparable with that of tRNA. However, its reactivity with N-OAc-2-AAF and enzymatically generated N-acetoxy-2-aminofluorene (N-OAc-2-AF) was only 20% of that of tRNA. Poly G was as reactive as tRNA toward synthetic N-OAc-2-AAF and enzymatically generated N-OAc-2-AF but was only 25% as efficient as tRNA or N-Ac-met in accepting enzymatically formed N-sulfonoxy-2-AAF. The difference in the interaction of the three nucleophiles with N-OAc-2-AAF and N-OAc-2-AF compared with N-sulfonoxy-2-AAF indicate that N-OAc-2-AAF is not a general model of the ultimate electrophilic metabolites of 2-AAF. Studies of the kinetics of the interaction of N-OAc-2-AAF with N-Ac-met, tRNA and poly G demonstrated dependence of adduct formation on nucleophile concentration, indicative of a bimolecular mechanism. Arylamidonium or nitrenium ions are therefore not necessarily the ultimate electrophilic metabolites of 2-AAF obligatory for interaction with cellular nucleophiles. There was no evidence that nitrenium ions are intermediates in the cytosolic reduction of N-OH-2-AAF to 2-AAF.

Peroxidase-catalysed binding of [U-14C]phenol to DNA.

14C-Phenol binds irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide, approximately 65% of the added phenol was bound to DNA. Binding was maximal at an equimolar concentration of hydrogen peroxide. Binding also occurred to homopolyribonucleotides polyadenylic acid, polyguanylic acid, polycytidylic acid and polyuridylic acid, and suggests that binding is relatively non-specific with respect to the nucleotide bases. p,p'-Biphenol, p,p'-biphenoquinone, o,o'-biphenol and two unidentified products were formed by the oxidation of phenol, in the presence and in absence of DNA. DNA accelerated phenol oxidation four fold and prevented the polymerization of oxidized phenol products, but was found to have no effect on the range of ethyl acetate-extractable products. Phenol accelerated the metabolism of o,o'-biphenol but had no effect on p,p'-biphenol metabolism. The mechanism of phenol activation is not clear, but p,p'-biphenoquinone binds to protein and not to DNA. DNA binding was prevented by glutathione, N-acetyl-cysteine and ascorbate, and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetyl-cysteine. DNA binding was not inhibited by lysine and proline.

Preparation and biological properties of a highly active poly(G) · poly(C) inducer of interferon

Experimental conditions necessary for the full expression of interferon-inducing activity by a complex of polyguanylic acid and polycytidylic acid included: (i) a sufficiently high molecular size of each homopolymer; (ii) annealing conditions which insured complete denaturation of polyguanylic acid self-structure; and (iii) the specific biological assay employed to assay interferon-inducing potency. The complex of polyguanylic acid and polycytidylic acid possessed several properties that suggested it may be an atypical polynucleotide interferon inducer. For instance, it was inactive in primary rabbit kidney cell cultures, usually exquisitely sensitive to polynucleotide interferon inducers, unless it was incubated on the cell cultures for prolonged times or in the presence of DEAE-dextran. Polyguanylic acid · polycytidylic acid could induce interferon in rabbits and mice but gave a more protracted response than did poly(I) · poly(C). Finally, poly(G) · poly(C) was, without any modification, resistant to degradation by serum nucleases.

Solubilization and reconstitution of a nuclear envelope-associated ATPase. Synergistic activation by RNA and polyphosphoinositides.

Treatment of isolated rat liver nuclear envelopes with 1% Triton X-100 solubilized 20-30% of the nuclear envelope protein and 85% of the ATPase activity. Chromatography on DEAE-Sepharose in 1% Triton X-100 at pH 7.5 removed all of the chemically measurable phospholipid from the bound ATPase activity; however, endogenously synthesized phosphatidylinositol [4-32P]phosphate (PIP) co-chromatographed with the ATPase activity on this column. Further purification by chromatography on heparin-agarose removed all of the [32P]PIP and RNA from the ATPase; however, the recovery of ATPase activity from this column was very low. RNA, polyadenylic acid, and polyguanylic acid stimulated the delipidated ATPase activity 4-6-fold. This activity was not further stimulated by the addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid, but was stimulated 2-fold by the addition of phosphatidylinositol. Addition of 40 microM PIP and 50 micrograms of RNA/ml resulted in a 25-fold stimulation of basal ATPase activity. Phosphatidylinositol 4,5-bisphosphate, in the presence of RNA, was also able to stimulate ATPase activity but to a lower extent than that of PIP. Therefore, the nuclear envelope-associated ATPase appears to require interaction with a polynucleotide and PIP to express full activity. PIP, which is actively metabolized in the nuclear envelope, may therefore be involved in the regulation of the activity of this enzyme.

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine.

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.

Metabolic activation of the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide by prostaglandin H synthase.

It has been demonstrated that N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), when fed with the diet, causes transitional carcinomas in rats. An important step in the mechanism of NFTA-induced carcinogenesis is endogenous metabolic activation to an ultimate carcinogen. We have proposed that the enzyme complex prostaglandin H synthase (PHS) is involved in the activation of certain renal and urinary tract carcinogens. This proposal was assessed by examining the activation of the 5-nitrofuran renal carcinogen NFTA and its deacetylated analogue 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) by PHS. Ram seminal vesicular and rabbit renal inner medullary microsomes were used as a source of PHS. Both NFTA and ANFT were activated by PHS to bind microsomal protein. Both microsomal preparations activated ANFT to bind DNA. However, only ram seminal vesicular microsomes activated NFTA to bind DNA. The rate of ANFT binding to macromolecules was considerably greater than NFTA with both microsomal preparations. Although activated ANFT was shown to bind several different homopolynucleotides, a preference for binding polyguanylic acid was demonstrated. Glutathione inhibition of carcinogen binding to macromolecules was shown to be due to the formation of a thioether conjugate. Deacetylation of NFTA was demonstrated in both tissues with deacetylation significantly exceeding acetylation of ANFT to NFTA in the kidney. Thus, renal PHS activation of both NFTA and ANFT was demonstrated with the rate of ANFT activation being considerably greater than NFTA. The conversion of NFTA to ANFT by intact tissue suggests that ANFT may contribute to NFTA renal carcinogenesis.

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts.

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.

Prostaglandin H synthase-mediated reaction of carcinogenic arylamines with tRNA and homopolyribonucleotides

Prostaglandin H synthase mediates the reaction of an extensive series of carcinogenic arylamines with tRNA. Structure-activity relationships suggest that benzidine is especially reactive due to extended conjugation between the 4,4'-diamino groups. In trapping experiments with homopolyribonucleotides, benzidine reacts with polyguanylic acid but 4-aminobiphenyl reacts with polycytidylic acid. The nitrenium ion of 4-aminobiphenyl (formed by N,O-acyltransferase activation of N-hydroxy-4-acetylaminobiphenyl) reacts primarily with polyguanylic acid and to a lesser extent with polyadenylic acid. The results suggest that arylamine activation by prostaglandin H synthase does not involve nitrenium ion formation.

The effects of polyribonucleotides on the binding of dexamethasone-receptor complex to DNA

The ability of polyribonucleotides to affect the association of [ 3H]-Dexamethasone-receptor complex with DNA-cellulose has been investigated. Certain ribopolymers can release the receptor complex from DNA. This release is not dependent on the charge of the polymer only, but displays some specificity with regard to the base composition of the ribopolymer. Of the polyribonucleotides examined, polyguanylic acid and polyxanthylic acid were most effective at promoting release of the steroid-receptor from the DNA. Polyadenylic acid and polycytidylic acid had no effect on the association. For a polymer to display activity it must possess a base with an oxygen or sulfur atom at the C-6 position of purines or C-4 position of pyrimidines. Simple nucleotides possessing this structural feature are inactive.

Photoinduced reaction of dimethylnitrosamine with DNA and polynucleotides.

Abstract— The interaction between products from dimethylnitrosamine photolytic breakdown and DNA is time‐dependent and the reaction with polyguanylic acid is 36 times higher than that occurring with other polyribonucleotides; such interactions occur only if nucleic acids are present during dimethylnitrosamine photolytic breakdown. Among interaction compounds, neither O6‐methyl‐ nor 7‐methylguanine are observable; on the contrary, N‐methylhydrazine and trimethylamine are always found in acid and alkaline hydrolysates of DNA and polynucleotides. These findings are very similar to the results previously found in biological in vivo and in vitro systems and suggest a possible usefulness of this physicochemical system as an analogical model of chemical carcinogenesis.

Effect of polynucleotides on the dimerization of glycine.

Polynucleotides were found to suppress the dimerization reaction of aqueous glycine with trimetaphosphate as the condensing agent. Small anions (chloride, acetate, and phosphate) did not show this effect. The reaction was studied at a pH of about 11.5 and at 70 degrees C and room temperature with a 13 mM concentration of glycine and trimetaphosphate. Under these conditions, the effect of the polynucleotides was in the following order: polyguanylic acid less than polycytidylic acid less than polyadenylic acid less than polyuridylic acid. The result may have a significant implication for the understanding of processes of chemical evolution.

Characterization of excitation spectra for Tb 3+ luminescence from nucleic acids: Calcium binding environs in icosahedral viruses

This communication characterizes the excitation spectra of enhanced Tb 3+ emission at 545 nm due to energy transfer from nucleic acid bases. For 5′GMP and polyguanylic acid the excitation spectrum maximum occurs at 252 nm and, for polycytidylic acid, at 270 nm. Both in their maxima and general shape the excitation spectra correspond closely to the absorption spectra of the aromatic chromophores. To obtain reliable excitation spectra it is necessary both to have solutions of low absorbance in order to avoid inner-filtering of incident radiation and to correct for the severe decrease of xenon lamp intensity at < 270 nm. In samples of yeast and E. coli t-RNA, guanine bases are primarily responsible for enhanced Tb 3+ emission. The two icosahedral viruses, bromegrass mosaic virus and southern bean mosaic virus, display excitation spectra for Tb 3+ emission similar to that of the t-RNA samples. In the viruses, energy transfer to Tb 3+ in a presumed Ca 2+ site originates primarily from guanine bases and not from aromatic chromophores in the protein component.

Partial Characterization of Ribonuclease (RNase) Activity from the Isolated and Solubilized Brush Border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.

Microsomal activation of thioacetamide- S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [ 3H]thioacetamide- S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O 2 or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide- S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide- S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [ 3H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide- S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.

Comparison of commercial reversed-phase high-performance liquid chromatographic columns for the separation of benzo[ a]pyrene diolepoxide-nucliec acid adducts

The separation of benzo[ a]pyrene diolepoxide-nuicleoside and deoxynucleoside adducts on μ-Bondapak C 18, Radial-Pak C 18, Partisil ODS-2, Micropak MCH-5 Zorbax ODS and Ultrasphere ODS high-performance liquid chromatographic columns has been compared under identical solvent conditions. The nucleoside and deoxy-nucleoside adducts were obtained by chemical and enzymatic digestion of the nucleic acid products of the reactions of benzo[ a] pyrene diolepoxides with polyguanylic acid and DNA. Significant differences in separation were observed with the columns tested which could not be correlated with particle size, carbon content, surface area, or surface coverage. Baseline separation of eight diolepoxide-guanosine adducts was obtained on the Ultraphere ODS column. The separation of the diolepoxide- deoxynucleoside adducts on the Ultrasphere ODS column suggests that it will be useful in identifying new benzo[ a]pyrene DNA adducts.

Arachidonic acid-dependent metabolism of (±)-7,8-dihydroxy-7,8-dihydrobenzo[A]pyrene to polyguanylic acid-binding derivatives

Microsomal and solubilized enzyme preparations from ram seminal vesicles catalyze the oxidation of (±)-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene (BP-7,8-diol) to derivatives which covalently bind to polyguanylic acid (poly(G)). Oxidation requires prostaglandin endoperoxide synthetase and its substrate arachidonic acid and is inhibited by indomethacin. High performance liquid chromatographic (HPLC) analysis of the nucleoside adducts obtained following chemical and enzymatic digestion reveals an adduct pattern which is identical to that obtained by the reaction of poly(G) with (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (diol-epoxide 2). The ratios of the diastereomeric pairs of cis- and trans-diol-epoxide 2-guanosine adducts indicate that both enantiomers of (±)-BP-7,8-diol are metabolized to an equal extent.