Search

The fungus, Cunninghamella elegans, was used as a microbial model of mammalian drug metabolism to biotransform a tricyclic antidepressant, cyclobenzaprine. Seventy-five percent of this drug at a concentration of 1 mM was metabolized within 72 h by C. elegans grown on Sabouraud dextrose broth. Milligram amounts of fungal metabolites were isolated by reversed-phase high performance liquid chromatography (HPLC) and their structures were characterized by 1H NMR spectroscopy, mass spectrometry, and UV spectroscopy analyses. The major fungal metabolites of cyclobenzaprine were 2-hydroxycyclobenzaprine (59%), N-desmethylcyclobenzaprine (21%), cyclobenzaprine trans-10,11-dihydrodiol (5%), N-desmethyl-2-hydroxy-cyclobenzaprine (3%), 3-hydroxycyclobenzaprine (3%), and cyclobenzaprine N-oxide (1%). These fungal metabolites were used as standards to investigate the metabolism of cyclobenzaprine by rat liver microsomes. Rat liver microsomes also biotransformed cyclobenzaprine to produce similar metabolites as the fungus. The isotope labeling of 2-hydroxycyclobenzaprine by 18O2 and the trans-configuration of the dihydrodiol suggested that these reactions were catalyzed by cytochrome P-450 monooxygenases in C. elegans. These results also demonstrated that the fungal biotransformation system could be used to predict and synthesize the mammalian drug metabolites.

1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals. Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-yl)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene. In this paper, we report the identification of two N2-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-yl)-1-aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA. These DNA adducts were identified as 6-(deoxyguanosin-N2-yl)-1-aminopyrene and 8-(deoxyguanosin-N2-yl)-1-aminopyrene. The two N2-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitrosopyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene. The DNA adducts were also formed when 1-nitropyrene was metabolized by xanthine oxidase in the presence of calf thymus DNA, and when 1-nitropyrene was activated by rat liver microsomes and cytosols, as well as from DNA isolated from Salmonella typhimurium suspension cultures incubated with 1-nitropyrene.

Pure cultures of anaerobic intestinal bacteria and mixed fecal microflora from human, rat, mouse, and pig were screened for the ability to metabolize 7‐nitrobenz[a]anthracene (7‐NO2BA). Based on analysis by high‐performance liquid chromatography (HPLC) and by ultraviolet (UV), mass, and nuclear magnetic resonance (NMR) spectral techniques, the compounds were identified as 7‐aminobenz[a]anthracene (7‐NH2BA) and benz[a]anthracene 7, 12‐dione (dione). Identification of 7‐NH2BA as a metabolite of 7‐NO2BA indicates that the anaerobic intestinal bacteria are capable of reducing 7‐NO2BA to potentially bioactive intermediates. The reductive capacities of the mixed intestinal microflora were generally greater than those of pure cultures. Thus, metabolism of 7‐NO2BA in the intestinal tract may 6e underestimated if pure cultures are used as the sole method for evaluating the potential hazard.

We investigated the metabolism of 3-nitrofluoranthene by filamentous fungus, Cunninghamella elegans ATCC 36112. Cunninghamella elegans metabolized about 72% of the 3-nitro[3,4-14C]fluoranthene added during 144 h of incubation to 2 major metabolites. These metabolites were separated by reversed-phase high-performance liquid chromatography and identified as 3-nitrofluoranthene-8-sulfate and 3-nitrofluoranthene-9-sulfate by 1H nuclear magnetic resonance, UV-visible, and mass spectral techniques. These results, in conjunction with previous studies on the fungal metabolism of fluoranthene, indicate that the nitro substituent at the C-3 position of fluoranthene sterically hinders epoxidation and shifts metabolism to the C-8 and C-9 positions. Since the phenolic microsomal metabolites of 3-nitrofluoranthene are mutagenic, the formation of sulfate conjugates of 8- and 9-hydroxy-3-nitrofluoranthene by C. elegans suggests that the fungal metabolic pathways may be beneficial for detoxification of this ubiquitous pollutant.

A metabolite of benzo[a]pyrene, 9-r,10-t-dihydroxy-7,8-c-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-III), that is not thought to be involved in carcinogenesis has nevertheless been shown to bind extensively to DNA in vitro. The adducts formed by this non-bay-region diol epoxide in Chinese hamster ovary cells are much less mutagenic than those formed by an isomeric diol epoxide that is carcinogenic. We have isolated and characterized three major adducts formed by in vitro reaction of BPDE-III with DNA. The major adduct, accounting for over half of the total is formed by reaction of BPDE-III with the N7 position of dGuo and is recovered after enzymatic digestion as an N7-Gua adduct. A second major adduct involves the N2 position of dGuo, while the third adduct is tentatively identified as a C8-substituted dGuo. Little or no reaction with deoxyadenosine residues is detected. The N7 adduct is unstable in DNA at 37 degrees C and is released as the modified base with a half-life of about 24 h. This adduct lability apparently leads to single-strand breaks and alkali-sensitive sites in the DNA and may account in part for some of the biological properties of BPDE-III adducts. This represents the first description of an N7-dGuo adduct that is formed in DNA as the major adduct by a diol epoxide derived from a carcinogenic polycyclic aromatic hydrocarbon.

Natural abundance 15N NMR spectroscopy and ancillary spectroscopic techniques have been employed to study the solution structure of 8-hydroxyadenosine. 8-Hydroxyadenosine is a naturally occurring oxidized nucleic acid adduct that is generally implied to have an 8-hydroxy tautomeric structure. 15N NMR chemical shifts and coupling constants, however, indicate that the modified base exists as an 8-keto tautomer. The pH dependence of 15N NMR and UV spectra showed the presence of two pKa's, at 2.9 and 8.7, corresponding to protonation at N1 and ionization at N7, respectively. The latter results in the formation of an 8-enolate structure. Unusual upfield shifts of the 1H and 15N resonances of the NH2 group, and a reduction in the one-bond coupling constant 1JN6-H6, is indicative of an unfavorable steric or electronic interaction between the NH2 group and the adjacent N7-H proton. This interaction results in a subtle change in the structure of the NH2 group. In addition to being a possible mechanism for alteration of hydrogen bonding in oxidized DNA, this type of interaction gives a better understanding into N7-N9 tautomerism of adenine. Furthermore, the structure of 8-hydroxyadenosine has been related to possible mechanisms for mutations.

2-Nitropyrene, the geometric isomer of the most studied nitropolycyclic aromatic hydrocarbon (nitro-PAH), 1-nitropyrene, is an environmental contaminant detected in ambient air and a potent direct-acting mutagen. Its metabolic activation leading to the formation of DNA adducts was studied. The activated metabolite, N-hydroxy-2-aminopyrene, was prepared and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA, the resulting nucleosides were separated by HPLC, and the adducts were characterized by mass and proton NMR spectral analysis. Both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene, in a 5:2 ratio, were identified. These adducts were then utilized as standards to identify the DNA adducts formed from reaction of [3H]2-nitropyrene with DNA mediated by liver microsomes and cytosols of mouse and rat. In all cases, both adducts were formed. The quantities of the two adducts formed in each system were: mouse liver microsomes (11.3 pmol [3H]2-nitropyrene/mg DNA), rat liver microsomes (23), mouse liver cytosol (11.4) and rat liver cytosol (5.1). Thus, these adducts were formed in highest yield from rat liver microsomes and the lowest from rat liver cytosol. The deoxyguanosine/deoxyadenosine adduct ratio was higher from rat and mouse liver microsomes (7.8:9.2) than from rat and mouse liver cytosols (2.5:3.1). Our results represent the first direct demonstration of a C8-deoxyadenosine adduct being formed as a major product from the reaction of a nitro-PAH metabolite with DNA.

Quinoxaline, a mutagenic azaarene produced in foods during cooking, was added to cultures of Streptomyces badius ATCC 39117. After 24 h, the cultures were extracted with ethyl acetate. Two major metabolites were purified by liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy as 3,4-dihydro-2(1H)-quinoxalinone and 2(1H)-quinoxalinone.

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterial mutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P, prepared in situ from reduction of 3-nitro-B[a]P with calf thymus DNA, was studied. After enzymatic digestion of the DNA, the resulting modified nucleosides were analyzed by thermospray HPLC-MS and high-resolution proton NMR spectroscopy. The major adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubation of DNA with 3-nitro-B[a]P in the presence of the mammalian nitroreductase xanthine oxidase, and hypoxanthine. These data indicate that a mammalian nitroreductase can metabolize 3-nitro-B[a]P to an activated derivative that reacts with DNA to give a novel adduct distant from the site of N-hydroxylation.

The environmental contaminants 1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are mutagens in Chinese hamster ovary (CHO) cells with exogenous metabolic activation. Previous studies demonstrated the potent direct-acting mutagenicity of the oxidized metabolites, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-NBaPDE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (3-NBaPDE), and the partially nitroreduced metabolites, 1- and 3-nitrosobenzo[a]pyrene (1- and 3-NO-BaP). In this study, we have identified the major adduct formed by incubation of calf thymus DNA with 1-NBaPDE and used this standard in conjunction with other adduct standards to characterize the 32P-postlabeled DNA adducts produced by 1- and 3-nitro-BaP metabolites in CHO cultures. The major adduct from 1-NBaPDE exposure was 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene; from 3-NBaPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene; from 1-NO-BaP, 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene; and from 3-NO-BaP, 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene. For comparison, the adducts formed by trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the related nitroreduced derivative 6-nitrosobenzo[a]pyrene were also examined. The nitrobenzo[a]pyrene DNA adducts described in this study are proposed to be involved in the mutagenicity of 1- and 3-nitro-BaP upon either oxidative or reductive metabolism.

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are direct-acting mutagens and carcinogens that are considered a risk to human health. We investigated the metabolism of 2-nitrofluorene by the fungus Cunninghamella elegans ATCC 36112. At 144 h of incubation, C. elegans had metabolized about 81% of the [9-14C]-2-nitrofluorene, resulting in 6 metabolites. The major metabolites were separated by reversed-phase high-performance liquid chromatography and identified by 1H NMR, ultraviolet (UV)-visible, and mass spectral analyses as 2-nitro-9-fluorenol, 2-nitro-9-fluorenone, 6-hydroxy-2-nitrofluorene, and sulfate conjugates of 7-hydroxy-2-nitro-9-fluorenone and 7-hydroxy-2-nitrofluorene. 2-Nitro-9-fluorenol accounted for about 62% of the total metabolism. For comparison with the microbial system, experiments with liver microsomes of rats pretreated with 3-methyl-cholanthrene were conducted. Microsomal incubations indicated formation of phenolic and ring-hydroxylated products of 2-nitrofluorene. 2-Nitrofluorene and hydroxylated metabolites have been previously implicated as direct-acting mutagens in bacterial assays and have shown sister chromatid exchanges in vivo in bone marrow cells and in vitro in ovary cells and unscheduled DNA synthesis in mammalian studies. Previous studies with other PAHs using C. elegans have shown that the phenols and glucoside and sulfate conjugates of phenols are generally less mutagenic than the parent. The results from the metabolism of 2-nitrofluorene by C. elegans suggests the detoxification potential of this fungus.

3-Aminochrysene, a mutagenic geometric isomer of the mutagenic and carcinogenic aromatic amine 6-aminochrysene, has been synthesized and its metabolic activation studied by characterization of the products formed from the reaction of metabolites with calf thymus DNA. DNA adducts produced by 3-aminochrysene via N-oxidation were examined by preparing 3-nitrosochrysene and incubating the nitroso derivative with calf thymus DNA in the presence of ascorbic acid (to generate the N-hydroxy derivative) at pH 5. The major adduct, as determined by 1H-NMR and thermospray-mass spectrometry of the modified nucleoside obtained after enzymatic hydrolysis of the modified DNA, was N-(deoxyguanosin-8-yl)-3-aminochrysene. Thus, the reaction of N-hydroxy-3-aminochrysene with DNA differs from that of N-hydroxy-6-aminochrysene, which had previously been shown to generate N-(deoxyguanosin-8-yl)-6-aminochrysene, 5-(deoxyguanosin-N2-yl)-6-aminochrysene and N-(deoxyinosin-8-yl)-6- aminochrysene as major adducts. 32P-Postlabeling analysis of DNA treated with 3-aminochrysene in the presence of liver microsomes from rats pretreated with phenobarbital indicated an adduct pattern identical to that seen with DNA that had been treated with 3-nitrosochrysene and ascorbic acid. However, DNA treated with 3-aminochrysene (3-AC) in the presence of liver microsomes from rats pretreated with 3-methylcholanthrene contained a major adduct that was chromatographically distinct from N-(deoxyguanosin-8-yl)-3-aminochrysene.

The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.

Analyses of high resolution proton and carbon NMR spectra of a series of guanine nucleosides in DMSO have revealed a near linear correlation between the chemical shift of the H2, atom of the sugar moiety and the vicinal coupling constant 3JC4-H1'. This unexpected result provides evidence that the variations in the glycosyl torsion angle between nucleosides in solution are less that those which have previously been reported in crystals and it is an experimental basis for analyzing the syn and anti populations from chemical shift and coupling constant data.

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high-pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed-phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N-(2-methoxyphenyl)-3-oxobutanamide (o-acetoacetanisidide), 2-(hydroxyimine)-N-(2-methoxyphenyl)-3-oxobutanamide and N,N''-bis(2-methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [14C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of 18O2 led to the isolation of 1-naphthol that contained 94% 18O. The metabolism of [1-2H]- and [2-2H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-2H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The 18O2 and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.

L'analyse de la conformation et de la dynamique associee a la liaison aryl-azote et amide pour les acetylamino-2 fluorene (AAF) ainsi que ses derives N-hydroxy, N-acetoxy et N-sulfate est realisee comme l'etape initiale pour l'etude de l'orientation de AAF dans les acides nucleiques modifies. Discussion des resultats des spectres RMN de 1 H, 13 C et 15 N

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E. coli glycosylase. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.

Nuclear magnetic resonance spectra of 5'-AMP were examined over a concentration range of 0.001 to 2.2 m in the Fourier mode in the 1H and 1H-{31P} configuration and were analyzed by computer simulation. The principal conclusions with respect to the solution conformation of 5'-AMP are as follows. (a) The exocyclic C(5')—O(5') and C(4')—C(5') bonds are flexible, and preferentially exist in the gauche-gauche conformation with a "W" relationship across H(4')—C(4')—C(5')—O(5')—P(5') in which the above atoms lie in one plane as in its crystal structure. (b) The ribose moiety exists as a flexible ring system undergoing interconversion between the 3E and 2E conformations. The sugar ring conformation is strongly concentration dependent, being 63 ± 10% 2E and 37 ± 10% 3E at concentrations below 0.01 m. At higher concentrations, the population of the 3E form increases at the expense of the 2E form, and at 2.0 m, 3E is the preferred conformation. In the solid state the conformation of the sugar ring is 3E for 5'-AMP.

The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.

Summary The 300 MHz 1 H and 40.48 MHz 31 P NMR spectra of ApA, dApdA, and several purine nucleotides in aqueous solution have been completely analyzed at various temperatures or concentrations, and the derived coupling constant data interpreted in terms of dynamic conformational parameters by means of a Karplustype analysis. It is found that base stacking interactions play a key role in reducing conformational flexibility. The C(4′)-C(5′) and C(5′)-o(5′) bonds in both oxy- and deoxy- adenyl dinucleosides become increasingly gauche conformation with increasing base stacking, while the ribose ring in ApA correspondingly becomes more 3 E conformation and the deoxyribose ring in dApdA remains mostly 2 E conformation.

Purine ring-opened 7-methylguanine, prepared in vitro by alkaline treatment of 7-methylguanosine or of methylated calf thymus DNA, was extensively characterized by chromatographic and spectral techniques as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine. This modified base chromatographed as an early-eluting peak on an ion-exchange column but separated into two interconvertible components after reversed-phase or porous-resin h.p.l.c. The two components were analyzed by thermal desorption mass spectrometry and 500 MHz 1H-n.m.r. spectroscopy. Their mass spectra were identical (M+ at m/z 183) and their n.m.r. spectra each exhibited the same two sets of resonances whose relative intensities were solvent-dependent. Analysis by h.p.l.c. showed interconversion of the two components and kinetic studies demonstrated that this reaction was a reversible first-order process. At equilibrium, k1 = k2 = 0.334 h-1 and delta G = 22.9 kcal/mol. These data indicated that the ring-opened 7-methylguanine exists as cis/trans isomers with restricted rotation about the amide bond. Treatment of rats with an intraurethral initiating dose of the carcinogen N-methylnitrosourea resulted in a high level of bladder epithelial DNA modification with 7-methylguanine, O6-methylguanine, and methyl phosphotriesters as major adducts at 2 h after instillation. Purine ring-opened 7-methylguanine, chromatographically identical to the in vitro products, was initially a minor adduct. However, it was the only persistent modification in the bladder epithelial DNA and eventually accounted for 72% of the total carcinogen binding after 21 days. A tumor-promoting regimen, involving dietary sodium saccharin, did not alter the repair or persistence of any of the methylated adducts. These data demonstrate that purine ring-opened 7-methylguanine, previously reported to exist in liver DNA after N,N-dimethylnitrosamine or 1,2-dimethylhydrazine treatment, is present in a carcinogen-target tissue and is considerably more persistent than O6-methylguanine or other DNA methylation products. The possible role of this adduct as a promutagenic lesion initiating urinary bladder carcinogenesis is discussed.

The solution conformation associated with the fluorenyl nitrogen atom of 8-(N-fluoren-2-ylacetamido)-2′-deoxyguanosine 5′-monophosphate was investigated by 500 MHz 1 H and 67.89 MHz 13 C nuclear magnetic resonance spectroscopy. Barriers of 12–15 kcal/mole to internal rotation about the guanyl-nitrogen bond and the aryl amide bond were found. The energy minima conformations about the guanyl-nitrogen bond were characterized by an approximate orthogonal orientation between the planes of the acetamido moiety and the guanine ring. The four torsional diastereomers interconvert through a cyclic pathway. The torsional barriers are of importance because they may be a factor altering normal base stacking in modified DNA.

The conformation of the sugar moiety of 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine in solution has been examined as a function of temperature by 1H-nmr spectroscopy. Analysis of coupling constants shows that lowering the temperature to −50°C in methanol shifts the conformational equilibrium of the sugar ring resulting in a C2′-endo conformation at a mole fraction of 0.97. The computed phase angle of pseudorotation and amplitude of pucker are 154° and 36°, respectively, with very little discrepancy between the five calculated coupling constants and coupling constants extrapolated from the temperature profiles. A computer program has been written enabling a three-state best-fit analysis. The three-state analysis indicates an equilibrium between C2′-endo, C3′-endo, and 04′-endo conformations. In aqueous solution, the computed mole fraction of the 04′-endo form is 0.18 at 30°C. The conformation associated with the sugar ring and the C4′C5′ bond is compared to that of 2′-deoxyguanosine.

The 40-MHz 31P nuclear magnetic resonance (nmr) spectrum of intact HeLa cells contains seven broad peaks with some detectable splittings. The linewidths were significantly broader than for those of cell-free systems such as cell extracts, indicating that the cellular environment is responsible for the unusual line broadening. Resolution of these peaks at 40 MHz is sufficient to make certain assignments and the relaxation parameters of some of the intracellular metabolites have been measured. The spin-lattice relaxation times (T1) ranged from 0.3 s for adenosine triphosphate (ATP) to about 3 s for inorganic phosphate (Pi) and monophosphate compounds. Nuclear Overhauser enhancements (NOE) were induced by proton irradiation with the possible exception of ATP. The relaxation parameters were compared to those of cell-free compounds and in all cases T1 and NOE were smaller for the intracellular metabolites. The relaxation parameters for ATP were affected the most. This behavior was mimicked with mixtures of cell-free metabolites containing paramagnetic ions. The larger change in both T1 and NOE of intracellular ATP could be accounted for by selective binding of paramagnetic ions. This phenomenon also explains some of the line broadening in the cell spectrum especially that of ATP. The spin-spin relaxation times (T2) of P1 and monophosphate compounds as measured by a pulse technique did not account for the observed linewidths. This is due to the presence of chemical shift envelopes arising from pH heterogeneity. All resonances were broader at 146 MHz because of the line broadening by paramagnetic ions and the presence of chemical shift envelopes. Other mechanisms of line broadening may also be significant. There was little difference in resolution of spectra at 40 and 146 MHz. Water proton linewidths and T2 values were measured for HeLa cells and for some minced tissue preparations. The water linewidth in tissue samples was broader than that in the cell suspension. The large linewidths in tissues arise mainly from chemical shift envelopes caused by magnetic field nonuniformity in the tissue samples. There appears to be a small chemical shift envelope from magnetic nonuniformity in HeLa cells as well. The 1H results on envelopes were extrapolated to 31P studies on cells and tissues. Possible methods for reducing linewidths arising from the various proposed broadening mechanisms were discussed.

The probable ultimate urinary bladder carcinogen, N-hydroxy-2-naphthylamine (N-HO-2-NA), reacted with nucleic acids and proteins under mildly acidic conditions (pH 5) to form covalently bound derivatives. The extent of reaction was in the order: polyguanylic acid > DNA > or = protein > rRNA > tRNA > polyadenylic acid, polyuridylic acid > polycytidylic acid. At pH 7, appreciable reaction occurred only with protein. Enzymatic hydrolyses of the DNA, which contained 1.5 naphthyl residues/1000 nucleotides, yielded 3 nucleoside-arylamine adducts. From chemical, u.v., n.m.r., and mass spectrometric analyses, the adducts were identified as 1-(deoxyguanosin-N2-yl)-2-naphthylamine, 1-(deoxyadenosin-N6-yl)-2-naphthylamine, and a purine ring-opened derivative of N-(deoxyguanosin-8-yl)-2-naphthylamine, tentatively identified as 1-[5-(2,6-diamino-4-oxopyrimidinyl-N6-deoxyriboside)]-3-(2-naphthyl)urea. The properties of these adducts and their possible role in the initiation of carcinogenesis are discussed.

Enzyme binding studies have been conducted on several reduced nicotinamide adenine dinucleotide analogues having different substitutions at the 8 position of the adenine. The following analogues were synthesized for this study: 8-bromo-, 8-(methylamino)-, 8-(dimethylamino)-, and 8-(ethylamino)-substituted NADH. The conformation of these analogues was also studied. 1H and 13C nuclear magnetic resonance analysis showed that there was rotation about the adenine glycosyl bond and that the rotational preference depended on the C8 substituent. The bromo and dimethylamino analogues were predominantly in the syn conformation, while the anti conformation prevailed in the other derivatives as it does in the native NADH. Use of these analogues as co-enzymes by Pseudomonas aeruginosa transhydrogenase, Beneckea harveyi FMN:NADH oxidoreductase, rabbit muscle lactate dehydrogenase, beef heart lactate dehydrogenase, horse liver alcohol dehydrogenase, and yeast alcohol dehydrogenase resulted in enzyme activity in all cases. The bromo and dimethylamino analogues were bound significantly tighter than the other analogues for at least two of the enzymes studied. The data are discussed with respect to the ability of these enzymes to bind nucleotides which are in the syn conformation.

Proton magnetic resonance spectra of 5′AMP were taken in the concentration range of 0.001–2.2M. The concentration profiles of all the nonexchangeable protons were determined. The data for 5′AMP was compared to those of adenine, adenosine, and poly(A). Theoretically computed isoshielding lines of the adenine moiety were used to qualitatively predict a preferred stacking geometry of 5′AMP in aqueous solution. It is concluded that 5′AMP at pH 8 forms multistacked aggregates at high concentration levels and that a preferred orientation is such that the bases are aligned face to back with considerable, though less than 100%, base overlap; and that the ribose moieties of adjacent molecules are near one another with the phosphate groups well separated. Mn(II) ion binding studies show that the stacks are not restricted to one unique orientation type. Specific evidence is given showing that base-stacking orientations in the solid state may in some cases be considerably different from that in aqueous solution, due in part to numerous hydrogen bonding differences, and this is shown to be the case for base-stacked adenosine. In the case of 5′AMP the stacking orientations between the solid and liquid states are also different, except in this comparison the solid-state structure carries a positive charge.

Metabolism of 6-nitrobenzo[a]pyrene by rat liver microsomes yielded 1- and 3-hydroxy-6-nitrobenzo[a]pyrene, 6-nitrobenzo[a]pyrene-1,9- and -3,9-hydroquinone and benzo[a]pyrene-3,6-quinone. The monohydroxylated metabolites were more mutagenic than 6-nitrobenzo[a]pyrene in a Salmonella typhimurium / microsome reversion assay. These results indicate that ring hydroxylation is involved in the metabolic activation of this nitro-polycyclic aromatic hydrocarbon.

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)

The rat liver microsomal metabolism of 3-deutero-6-nitrobenzo(a)pyrene ([3-2H]6-NO2-BaP) was studied. The metabolites were separated by h.p.l.c. The 500 MHz 1H n.m.r. spectral analysis of the metabolites indicated that the 3-hydroxy-6-NO2-BaP and 6-NO2-BaP-3,9-hydroquinone each retained 33% of the deuterium label at the C-2 position. It is proposed that the migration of deuterium occurs via an NIH shift mechanism. These results indicate that a 2,3-epoxide is a common intermediate.

Aerobic metabolism of 1-nitrobenzo[e]pyrene (1-nitro-BeP) by rat liver microsomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP, and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichloropropylene 1,2-oxide was incorporated into the metabolism, 1-nitro-BeP 4,5-oxide was the predominant metabolite, and 1-nitro-BeP trans-4,5-dihydrodiol was not detected. All of the metabolites were purified by both reversed- and normal-phase HPLC and characterized by analysis of their mass and 500 MHz proton NMR spectral data. 1-Nitro-BeP was not metabolized under hypoxic conditions. 1-Nitro-BeP and its four metabolites were assayed in Salmonella typhimurium tester strains TA98, TA98NR and TA98/1,8-DNP6, both in the presence and absence of S9 activation. As predicted, 1-nitro-BeP was a weak mutagen without S9 (2 revertants/micrograms in TA98); the addition of S9 resulted in approximately 18, 17 and 4 revertants/micrograms in TA98, TA98NR and TA98/1,8-DNP6 respectively. The two phenolic metabolites were mutagenic both in the presence and absence of S9, producing moderate responses (19-84 revertants/micrograms). In addition, while the 1-nitro-BeP 4,5-oxide was only weakly mutagenic in TA98 (6-14 revertants/micrograms), 1-nitro-BeP trans-4,5-dihydrodiol was unexpectedly potent (approximately 300 revertants/micrograms both with and without S9). These results indicate that microsomal epoxidation of 1-nitro-BeP followed by epoxide hydrolase-catalyzed hydrolysis of the resulting epoxide to the 1-nitro-BeP trans-4,5-dihydrodiol results in the most potent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP 4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic than the corresponding parent nitro-PAHs. Also, the lower S9-mediated mutagenicity of 1-nitro-BeP in TA98/1,8-DNP6 compared with TA98 indicates that the mutagenicity of 1-nitro-BeP is dependent upon nitroreduction and transesterification. Finally, we previously hypothesized that nitrated PAHs with their nitro substituents perpendicular or nearly perpendicular to the aromatic rings are very weak or nondirect-acting mutagens in Salmonella typhimurium tester strains. The results reported in this communication demonstrate that ring-oxidized derivatives of nitro-PAHs do not always follow this structure--mutagenicity correlation.

A survey of phosphorus compounds present in HeLa cells and their acid extracts has been carried out by 31 P nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled 31 P spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 μmol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD + , glucose 6-phosphate, β-D-fructose 1,6-bisphosphate, α-D-fructose 1,6-bisphosphate, ADP, α-glycerophosphorylcholine, and α-glycerophosphorylethanolamine. UTP made up ⅕ of the total nucleotide triphosphate content. The composition was compared to the 31 P spectrum of an extract from a human astrocytoma grown in athymic mice. The signal from P-containing macromolecules such as nucleic acids was not detected in the intact HeLa cell spectrum because of broad lines. Effects of the glycolysis inhibitor iodoacetic acid could be clearly shown in spectra of both the intact cell and the extract as buildup of fructose 1,6-bisphosphate at the expense of ATP, UTP, and creatine phosphate.

The sesquiterpene diol with antispasmodic properties, earlier isolated from Cymbopogon proximus, is shown to be identical with cryptomeridiol.