Frederick E. Evans

Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene.

2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AFI) and N-(glutathion-S-yl)-2-aminofluorene (GS-AFII). Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AFI was derived from reaction with NOF and that GS-AFII could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AFI or be reduced to GS-AFII. The latter could also be formed by direct reaction with N-HO-AF.

Metabolism of the mutagenic environmental pollutant, 6-nitrobenzo[a]pyrene: Metabolic activation via ring oxidation

Abstract 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.

Photodecomposition of Pigment Yellow 74, a Pigment Used in Tattoo Inks¶

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.

IDENTIFICATION OF N5-METHYL-N5-FORMYL-2,5,6-TRIAMINO-4-HYDROXYPYRIMIDINE AS A MAJOR ADDUCT IN RAT LIVER DNA AFTER TREATMENT WITH THE CARCINOGENS, N,N-DIMETHYLNITROSAMINE OR 1,2-DIMETHYLHYDRAZINE

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.

Stereochemistry and evidence for an arene oxide-NIH shift pathway in the fungal metabolism of naphthalene

The mechanism of naphthalene oxidation by the filamentous fungus, Cunninghamella elegans is described. C. elegans oxidized naphthalene predominately to trans-1,2-dihydroxy-1,2-dihydroxy-1,2-dihydronaphthalene. A trans configuration was assigned for the dihydrodiol by nuclear magnetic resonance (NMR) spectroscopy at 500 MHz which showed a large coupling constant (J1,2) of 11.0 Hz. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydronaphthalene to that formed by mammalian enzyme systems indicated that the fungal dihydrodiol contained 76% (+)-(1S,2S)-dihydrodiol as the predominant enantiomer. Other naphthalene metabolites formed by C. elegans were identified as 1-naphthol, 2-naphthol and 4-hydroxy-1-tetralone. Incubation of C. elegans with naphthalene and 18O2 indicated that the trans-1,2-dihydroxy-1,2-dihydronaphthalene contained one atom of molecular oxygen which indicated a monooxygenase catalyzed reaction while similar incubations with naphthalene and H182O indicated that the other oxygen atom in trans-1,2-dihydroxy-1,2-dihydronaphthalene was derived from water. Mass spectral analysis of the acid-catalyzed dehydration products of the dihydrodiol indicated that the naphthalene dihydrodiol forms via the addition of water at the C-2 position of naphthalene-1,2-oxide. Fungal metabolism of [1-2H]naphthalene yielded 1-naphthol which retained 78% of the deuterium. NMR analysis of the deuterated 1-naphthol indicated an NIH shift mechanism in which deuterium migrated from the C-1 position to the C-2 position. The above results indicate that naphthalene-1,2-oxide is an intermediate in the fungal metabolism of naphthalene and that the fungal enzymes are highly stereo-selective in the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene.

Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans.

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.

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus

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.

Fast atom bombardment mass spectrometry of nucleoside and nucleotide adducts of chemical carcinogens

Abstract The xenon fast atom bombardment mass spectra of the N-acetylfluorenyl derivative of both mono and dinucleotides are characterized by the protonated molecular ion. Fragmentation resulting in cleavage of the glycosidic bond provides structural information.

Structure analysis of proximadiol (cryptomeridiol) by 13C NMR spectroscopy

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

Formation of sulfate and glucoside conjugates of benzo[e]pyrene by Cunninghamella elegans

Abstract 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.