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Carboxylate and salicylic OH coordinate bonding as well as intramolecular and intermolecular hydrogen bonding of bis-3,5-diisopropylsalicylatozinc(II), [ZnII(3,5-DIPS)2], with Lewis bases were studied to determine mechanisms accounting for antioxidant reactivity of ZnII(3,5-DIPS)2. Apparent thermodynamic parameters: K eq, ΔS 0, ΔH 0, and ΔG 0 were determined for these equilibria with bonding of two molecules of dimethyl sulfoxide-d6 (DMSO) or ethyl acetate-d8 (EA) to the ZnII using NMR and FTIR. We conclude that addition of two equivalents of DMSO or EA to non-polar solutions of ZnII(3,5-DIPS)2 results in bonding of DMSO or EA to ZnII via sulfoxide or ester carbonyl oxygen atoms with ternary complex formation, leading to weakening of carboxylate and salicylic OH coordinate bonding to ZnII and strengthening intramolecular hydrogen bonding between protons of salicylic OH groups and carboxylate oxygens. Subsequent addition of two or three additional equivalents of DMSO or EA leads to intermolecular hydrogen ...

High-field 1H-NMR methodology for enantiomeric composition determination of the chiral drug propranolol utilizing a chiral solvating agent is reported. Optimal experimental conditions for the resolution of enantiomers were determined by studying the interaction of substrate concentration, chiral solvating agent concentration and temperature. The success of the method is based on the selection of a chiral solvating agent that has the following two characteristics. First, it possesses functional groups that are complimentary to those of the chiral substrate for significant interaction to occur. Second, it has a group of diamagnetic anisotropy near its stereogenic center for translating spatial environments of solute nuclei into different magnetic environments that are measurable by NMR spectroscopy. Optical purities were determined on the basis of the intensities of the methyl proton resonances. The analysis of synthetic enantiomeric mixtures of propranolol by the proposed NMR method resulted in assay values, which agreed closely with the known quantities of each enantiomer in the mixtures tested. The mean +/- SD recovery values for the (R)-(+)-enantiomer was 100.0+/-0.6% of added antipode (n = 7).

The metabolism of metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] by the fungus Cunninghamella elegans ATCC 36112 was determined. The six metabolites identified comprised 81% of the total [14C]-metolachlor metabolized by C. elegans. These metabolites were separated by reversed-phase high-performance liquid chromatography and identified by 1H nuclear magnetic resonance, UV, and atmospheric pressure chemical ionization (APCI) mass spectral techniques. Metabolites I and II were identified as stereoismers of 2-chloro-N-[2-ethyl-6-hydroxymethylphenyl)]-N-(2-hydroxy-1-methylethyl)acetamide. Metabolites III and IV have been tentatively identified as stereoismers of 2-chloro-N-[2-(1-hydroxyethyl)-6-methylphenyl]-N-(2-methoxy-1-methylethyl)acetamide. Metabolites V and VI were identified as stereoismers of 2-chloro-N-(2-ethyl-6-hydroxy-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide and 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-hydroxy- 1-methylethyl)acetamide, respectively. The fungus Cunninghamella elegans was able to biotransform metolachlor. Multiple site oxidation of metolachlor by C. elegans occurred predominantly by O-demethylation of the N-alkyl side chain and benzylic hydroxylation of the arylalkyl side chain.

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.

Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-3H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by 1H and 13C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental pollutants; some are mutagenic, toxic, and carcinogenic and remain a public health concern. We investigated the metabolism of mixtures of PAHs and a tetracyclic aromatic hydrocarbon, chrysene, by the filamentous fungus, Cunninghamella elegans ATCC 36112. Cunninghamella elegans metabolized a mixture of PAHs including the carcinogen benzo[a]pyrene, phenanthrene, fluoranthene, pyrene, and acenaphthene completely to hydroxylated intermediates within 24 h. The metabolites from the PAH mixtures were similar to those formed in earlier studies of individual PAH compounds. In separate experiments with chrysene, C. elegans metabolized about 45% of the [5,6,11,12-14C]chrysene added to cultures during 144 h incubation. The two major metabolites of chrysene were separated by reverse-phase high performance liquid chromatography and identified by ultraviolet–visible, mass spectral, and1H-nuclear magnetic resonance techniques as sulfate conjugates of 2,8-dihydroxychrysene and 2-hydroxychrysene. The two major metabolites accounted for about 33% of the total metabolism. The formation of sulfate conjugates of phenolic chrysene metabolites and glucoside conjugates and hydroxylated products of PAH mixtures by C. elegans may be a detoxification step, because these types of products are generally less toxic than the parent compound. Key words: polycyclic aromatic hydrocarbons, PAH mixtures, chrysene, Cunninghamella elegans, biotransformation, oxidation.

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.

We recently reported that the reaction of N-hydroxy-3-aminobenzo[a] pyrene with calf thymus DNA produced 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene as the predominant adduct. The deoxyguanosinyl group of this adduct resides at the C6 position, which is remote from the reaction site, the nitrenium ion. It is significant to determine if formation of this type of DNA adduct is general and whether or not adduct formation is due to an increase in the stabilization of the nitrenium ion by increasing aromaticity. Thus, reduction of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene, both chemically and enzymatically, followed by reaction with calf thymus DNA was investigated. DNA was isolated and enzymatically digested, and the resulting modified nucleosides were separated by HPLC. Upon spectral analyses by mass and proton nuclear magnetic resonance spectroscopy, 6-(deoxyguanosin-N2-yl)-1-amino-7,8,9,10-tetrahydrobenzo[a] pyrene, 6-(deoxyguanosin-N2-yl)-3-amino-7,8,9,10-tetrahydrobenzo[a]pyrene, and 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene were identified, respectively. The same DNA adducts were formed from xanthine oxidase-mediated reductive metabolism of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene in the presence of calf thymus DNA. Thee results indicate that formation of N2-deoxyguanosinyl adducts of this type is common and that increasing the aromaticity by increasing the number of aromatic rings is not a decisive factor in directing their formation.

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 have been interested in the structure-activity relationships of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), and have focused on the correlation of structural and electronic features with biological activities, including mutagenicity and tumorigenicity. In our studies, we have emphasized 1-, 2-, 3-, and 6-nitrobenzo[a]pyrenes (nitro-B[a]Ps) and related compounds, all of which are derived from the potent carcinogen benzo[a]pyrene. While 1-, 2-, and 3-nitro-B[a]P are potent mutagens in Salmonella, 6-nitro-B[a]P is a weak mutagen. In vitro metabolism of 1- and 3-nitro-B[a]P has been found to generate multiple pathways for mutagenic activation. The formation of the corresponding trans-7,8-dihydrodiols and 7,8,9,10-tetrahydrotetrols suggests that 1- and 3-nitro-B[a]P trans-7,8-diol-9,10-epoxides are ultimate metabolites of the parent nitro-B[a]Ps. We have isolated a DNA adduct from the reaction between 3-nitro-B[a]P trans-7,8-diol-anti9,10-epoxide and calf thymus DNA, and identified it as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-ni tro-B[a]P . The same adduct was identified from in vitro metabolism of [3H]3-nitro-B[a]P by rat liver microsomes in the presence of calf thymus DNA. A DNA adduct of 3-nitro-B[a]P formed from reaction of N-hydroxy-3-amino-B[a]P, prepared in situ with calf thymus DNA was also isolated. This adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubating DNA with 3-nitro-B[a]P in the presence of the mammalian nitroeductase, xanthine oxidase, and hypoxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The structures of selected phenolic metabolites of 2- and 3-nitrofluoranthene have been analyzed. 1H NMR spectral analysis at 500 MHz using one- and two-dimensional methods proves that the site of hydroxy substitution in two metabolites previously reported as 3-nitrofluoranthen-8-ol and 3-nitrofluoranthen-9-ol had been reversed. A third and previously unidentified metabolite is shown to be 3-nitrofluoranthen-7-ol. Complete analysis of NMR spectral data on 2- and 3-nitrofluoranthene enabled confirmation of the previously reported structures of 2-nitrofluoranthen-8-ol and 2-nitrofluoranthen-9-ol from derived chemical shift substituent effects. Chemical shift data suggest that the nitro group is not strictly coplanar with the aromatic ring system in solution and that metabolism at a distant site can alter the conformation about the C-N bond of the nitro group. Reported mutagenicity data are analyzed relative to imine quinone formation, chemical shift substituent effects, electronegativity effects, and conformation in an attempt to explain the large differences in mutagenicity between the C8 and C9 hydroxy isomers of 2- and 3-nitrofluoranthene.

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.

Mycelia of Cunninghamella elegans were incubated in liquid Sabouraud medium containing 56 HM acridine. After 3 days, the culture medium was extracted with ethyl acetate and two metabolites were pur...

Anomalous 1H NMR spectra have been obtained for two related carcinogen-nucleoside adducts in which a 1- or 3-aminobenzo[a]pyrene ring is bound from its C-6 position to the N−2 position of 2′-deoxyguanosine. NOESY and COSY measurements at low temperature in methanol-d4 revealed chemical exchange between at least two forms. Large chemical shift differences exist between subspectra for all corresponding protons of the nucleoside moiety, whereas all such chemical shift differences are small for the protons of the benzo[a]pyrene moiety. Thus, at intermediate rates of exchange, the nucleoside resonances are broadened beyond recognition, whereas the aminobenzo[a]pyrene resonances appear normal. This is linked with the large anisotropic ring-current field from the carcinogen ring system, and restricted internal rotation at the site of attachment of the carcinogen to the nucleic acid base. The possibility of an absence of resonances from a nucleoside moiety can complicate structure elucidation of unknown carcinogen-nucleoside adducts and related compounds, especially in trace analysis.

The fungus Cunninghamella elegans ATCC 36112 transformed 98.6% of [ 14 C]alachlor [2-chloro-N-methoxymethyl-N-(2,6-diethylphenyl)acetamide] added to Sabouraud's dextrose broth to four metabolites within 96 h. Metabolism occurred predominantly by benzylic hydroxylation of one of the arylethyl side chains. Metabolites were separated by reversed-phase high-performance liquid chromatography and identified by 1 H nuclear magnetic resonance, UV, and mass spectral techniques

Several nontoxic purinethiols have been shown to block the ability of the carcinogen 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to bind covalently to DNA in Chinese hamster ovary cells. Two of these compounds also block BPDE-induced tumorigenesis in a two-stage mouse skin carcinogenesis model. The suggested mode of action of the purinethiols is through scavenging the electrophilic carcinogen by way of covalent reaction with the purinethiol. In the present work, we demonstrate that a series of five purinethiols (2,6-dithiopurine, thiopurinol, 6-thioxanthine, 2-mercaptopurine, and 9-methyl-6-mercaptopurine) react covalently in vitro with BPDE. The adducts formed have been characterized by UV-visible spectroscopy, solvent partitioning, and NMR spectroscopy; they result from addition of the thiol moiety at the 10-carbon of BPDE. Studies of the effects of Tris buffer and temperature on product ratios at completion of reaction indicate that the two major reaction pathways, hydrolysis of the epoxide and adduct formation, do not share a common rate-determining step. This suggests that the reaction mechanism for adduct formation is through SN2 attack of the thiol moiety at the 10 position of BPDE. The activation energies for the reaction of 5-purinethiols with various combinations of substituents at the 2 and 6 positions are all very similar, implying closely similar transition states. For compounds with a low pKa (2,6-dithiopurine, 2-mercaptopurine, and 6-thioxanthine) the most important reactant at physiological pH is the thiolate anion. However, for compounds with pKa's above 8, the physiologically important reactions appear to be more complicated.

A Mycobacterium sp. previously isolated from oil-contaminated estuarine sediments was capable of extensively mineralizing the high-molecular-weight polycyclic aromatic hydrocarbon fluoranthene. A carboxylic acid metabolite accumulated and was isolated by thin-layer and high-pressure liquid chromatographic analyses of ethyl acetate extracts from acidified culture media. The metabolite reached a maximum concentration of approximately 0.65% after 24 h of incubation. On the basis of comparisons with authentic compound in which we used UV and fluorescence spectrophotometry and R f values, as well as mass spectral and proton and carbon nuclear magnetic resonance spectral analyses, the metabolite was identified as 9-fluorenone-1-carboxylic acid. This is the first report in a microbial system of a fluoranthene metabolite in which significant degradation of one of the aromatic rings has occurred.

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.

Ambiguities in the 13C NMR chemical shift assignments of 1-nitropyrene were resolved using direct and long-range two-dimensional heteronuclear chemical shift correlation experiments. The latter was modified with a bilinear rotational decoupling (BIRD) pulse sequence, which enabled detection of all three-bond long-range correlations.

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.

The favored tautomeric and ionic structures were examined for the oxidative DNA damage adduct 8-hydroxy-2'-deoxyguanosine and its RNA analogue 8-hydroxyguanosine by 15N NMR spectroscopy. In addition, 15N chemical shifts and coupling constants from 13 different guanine nucleosides, including a wide variety of C8 substitutions (OH, SH, Br, OCH2C6H5, OCH3, SCH3, and SO2CH3), have been analyzed with respect to their tautomeric structures. A -98.5-Hz proton-nitrogen coupling constant observed for the N7 resonance of 8-hydroxyguanosine in dimethyl sulfoxide was evidence for 8-keto substitution, which is contrary to the structure implied by the generally used nomenclature. The pH dependence of 15N NMR spectra of 8-hydroxyguanosine in aqueous solution showed downfield shifts of the N1 and N7 resonances that were greater than 50 ppm, which indicated the conversion from a neutral 6,8-diketo to a 6-enolate-8-keto (pKa1 = 8.6) and finally to a 6,8-dienolate structure (pKa2 = 11.7). There was no evidence of an 8-enol substituent in the absence of ionization. It is proposed that the syn conformation of these oxidized bases in duplex DNA and RNA can be further stabilized by abnormal hydrogen bonding or mispairing that involves N7-H. The combined data show that 15N NMR is a sensitive probe to examine tautomerism of the guanine ring system. The analysis indicates that the change from a single to a double bond for the C8 substituent, and the accompanying removal of the normal double bond between N7 and C8 on the imidazole ring system, has no detectable effect on the tautomerism at the N1-O6 site of the pyrimidine ring system for both the 8-keto and 8-thio substitutions. In addition, large differences in electronegativity of the C8 substituents do not alter the N1-O6 tautomerism.

Water-suppressed proton nuclear magnetic resonance (NMR) spectroscopy of plasma has been proposed by Fossel et al. (N Engl J Med 1986; 315:1369-76) as a technique for detecting malignant tumors. In their analysis, plasma samples from patients with cancer were clearly distinguished from those of normal subjects by measuring and averaging the methyl and methylene line widths of plasma lipoproteins in NMR spectrums. To evaluate this diagnostic procedure further, we collected and analyzed by NMR spectroscopy 145 samples of plasma from patients who served as controls, most of whom were undergoing orthopedic or cardiac surgery (n = 66); patients with a variety of untreated malignant tumors (n = 25) or treated malignant tumors (n = 18); and patients with hyperplastic or "premalignant" diseases, such as benign prostatic hyperplasia and ulcerative colitis (n = 36). All the samples were coded, and NMR spectroscopy was performed without knowledge of the patients' clinical status. There were no significant differences in the NMR line widths among the four study groups (P greater than 0.05 for all pairwise comparisons). The specificity and sensitivity of this method for distinguishing the control patients (mean line width [+/- SD], 44.0 +/- 7.4 Hz) from those with untreated cancer (43.8 +/- 6.9 Hz) were poor, with a false positive rate of 52 percent (34 of 66) and a false negative rate of 56 percent (14 of 25). Inverse correlations of line widths with age (P less than 0.01) and with the plasma triglyceride level (P less than 0.001) were detected. We conclude that NMR spectroscopy of plasma is not an accurate test for the detection of malignant tumors.

The metabolism of 9-methylanthracene (9-MA), 9-hydroxymethylanthracene (9-OHMA), and 9,10-dimethylanthracene (9,10-DMA) by the fungus Cunninghamella elegans ATCC 36112 is described. The metabolites were isolated by high-performance liquid chromatography and characterized by UV-visible, mass, and 1H nuclear magnetic resonance spectral techniques. The compounds 9-MA and 9,10-DMA were metabolized by two pathways, one involving initial hydroxylation of the methyl group(s) and the other involving epoxidation of the 1,2- and 3,4- aromatic double bond positions, followed by enzymatic hydration to form hydroxymethyl trans-dihydrodiols. For 9-MA metabolism, the major metabolites identified were trans-1,2-dihydro-1,2-dihydroxy and trans-3,4-dihydro-3,4-dihydroxy derivatives of 9-MA and 9-OHMA. 9-OHMA was also metabolized to trans-1,2- and 3,4-dihydrodiol derivatives. The absolute configuration and optical purity were determined for each of the trans-dihydrodiols formed by fungal metabolism and compared with previously published circular dichroism spectral data obtained from rat liver microsomal metabolism of 9-MA, 9-OHMA, and 9,10-DMA. Circular dichroism spectral analysis revealed that the major enantiomer for each dihydrodiol was predominantly in the S,S configuration, in contrast to the predominantly R,R configuration of the trans-dihydrodiol formed by mammalian enzyme systems. These results indicate that C. elegans metabolizes methylated anthracenes in a highly stereoselective manner that is different from that reported for rat liver microsomes.

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-Nitrobenzo [a] pyrene (3-nitro-BaP) is a potent mutagenic environmental contaminant, and its biological activities have been intensively studied. It is significant to prepare its reactive metabolites and the corresponding modified DNA adducts for biological studies. The synthesis of its oxidized prorimate metabolite trans-7,8-dihydroxy-7,8-dihydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-trans-7,8-dihydrodiol, 1), its oxidized ultimate metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-DE, 2), and the corresponding DNA adduct 10-(deoxyguanosin-N 2 -yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitrobenzo [a] pyrene is described

The metabolism of fluorene, a tricyclic aromatic hydrocarbon, by Cunninghamella elegans ATCC 36112 was investigated. Approximately 69% of the [9-14C]fluorene added to cultures was metabolized within 120 h. The major ethyl acetate-soluble metabolites were 9-fluorenone (62%), 9-fluorenol, and 2-hydroxy-9-fluorenone (together, 7.0%). Similarly to bacteria, C. elegans oxidized fluorene at the C-9 position of the five-member ring to form an alcohol and the corresponding ketone. In addition, C. elegans produced the novel metabolite 2-hydroxy-9-fluorenone.

Mycobacterium sp. strain PYR-1, previously shown to extensively mineralize high-molecular-weight polycyclic aromatic hydrocarbons in pure culture and in sediments, degrades fluoranthene to 9-fluorenone-1-carboxylic acid. In this study, 10 other fluoranthene metabolites were isolated from ethyl acetate extracts of the culture medium by thin-layer and high-performance liquid chromatographic methods. On the basis of comparisons with authentic compounds by UV spectrophotometry and thin-layer chromatography as well as gas chromatography-mass spectral and proton nuclear magnetic resonance spectral analyses, the metabolites were identified as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy-1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde, benzoic acid, phenylacetic acid, and adipic acid. Authentic 9-hydroxyfluorene and 9-fluorenone were metabolized by Mycobacterium sp. strain PYR-1. A pathway for the catabolism of fluoranthene by Mycobacterium sp. strain PYR-1 is proposed.

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.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.

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.

The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.

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 structure of an adduct between guanine and the carcinogen acetylaminofluorene has been examined in the solid state by X-ray crystallography, and in solution by NMR techniques. The observed conformations have been compared with predictions from energy calculations and their relevance to models of adducts with DNA has been examined.