The covalent binding of the N-acetoxy-, N-hydroxy-, and nitro derivatives of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to 2'-deoxyribonucleosides or DNA was investigated in vitro and in vivo. N-Acetoxy-PhIP reacted with deoxyguanosine (dG), but not with the other deoxyribonucleosides, to form N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), whose structure was determined by NMR and mass spectral analyses and by ultraviolet absorption and pH-solvent partitioning characteristics. While reaction of N-acetoxy-PhIP with calf thymus DNA at pH 5.0 yielded 5.38 +/- 1.16 nmol of bound PhIP residues/mg of DNA, N-hydroxy-PhIP gave only 0.13-0.23 nmol binding/mg of DNA under identical reaction conditions. Nitro-PhIP produced no detectable binding under these conditions. HPLC analysis of 1-butanol extracts of enzymatically hydrolyzed DNA that had been modified by N-acetoxy-PhIP in vitro showed a major adduct which coeluted with and had an ultraviolet absorption and a mass spectrum that were identical to that of authentic dG-C8-PhIP. 32P-Postlabeling analysis of DNA isolated from colon, pancreas, lung, heart, and liver of rats treated orally with PhIP revealed the presence of a major PhIP-DNA adduct. This adduct had chromatographic properties identical to that of the 32P-labeled bis(phosphate) derivative of dG-C8-PhIP and represented 35-45% of the total adducts.(ABSTRACT TRUNCATED AT 250 WORDS)
We have analyzed the role of chitin, a cellwall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and(ii) by disrupting CHS3, the C. albicans homolog of CSD2/ CALJ/DITIOI/KT12, a Saccharomyces cereviswe gene required for synthesis of '90% of the cell-wall chitin. A key step in the development of an antifungal drug is the determination of its efficacy. For practical reasons, much of the initial testing is done in culture (in vitro), where the test conditions differ markedly from the conditions encountered by a fungal pathogen during infection (in vivo). The limited ability of in vitro testing to accurately predict in vivo efficacy is illustrated by the azoles, which are highly effective in vivo despite modest fungicidal activity in vitro (1). Thus, it is possible that some fungal components are required for pathogenicity in vivo but not for growth in vitro. Possible virulence determinants include factors required for recognition and invasion of the host and for protection against host defense systems. Because the cell wall is involved in these processesthe wall protects the fungal cell from external injury and cell-wall components mediate adherence (2-4) and immune response (5-8)-changes in cell-wall structure and/or composition may affect virulence.We have begun to investigate the role of the cell-wall polysaccharide chitin in the virulence of Candida albicans. Chitin is found in all true fungi; therefore agents that inhibit chitin synthesis are potential broad-spectrum antifungal drugs. In the past decade, many of the genes involved in chitin synthesis have been isolated in Saccharomyces cerevisiae, and, more recently, the homologs of these genes have been identified in C. albicans (9-11).In S. cerevisiae, three chitin synthases have been detected (for review, see refs. 12 and 13). One of them, chitin synthase III, makes 90% of the cell-wall chitin. At least three genes, CSD2/CALl/DIT101/KT12, CSD4/CAL2, and CAL3, are required for this activity. Mutants lacking this enzyme are chitin-deficient and, consequently, are resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth by disrupting microfibril assembly (14, 15).Like S. cerevisiae, C. albicans has at least three chitin synthases (9-11). Recently, the C. albicans homolog of CSD2, designated CHS3, was cloned and sequenced (11). By analogy to S. cerevisiae, C. albicans CHS3 mutants should be chitindeficient and Calcofluor-resistant. In the present report, we show that chitin-deficient mutants of C. albicans obtained by two methods are significantly less virulent than the wild-type strain. . Media were supplemented with uridine at 0.1 mg/ml as required. Agar (2%, Difco) was added for solid media. Urdauxotrophs were selected on medium containing 5-fluoroorotic acid (5-FOA) as described (16). Germ-tube formation was induced in 20% fetal bovine serum ...
The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24°C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable '4C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cisand trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 1802 studies showed that the formation of cisand trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.
A recent epidemiological study suggested that aromatic amines present in hair dyes may contribute to an increased risk of bladder cancer (Gago-Dominguez, et al. (2003) Carcinogenesis 24, 483-489). Moreover, a preliminary study linked frequent hair dye usage with elevated levels of DNA adducts of 4-aminobiphenyl (4-ABP) in human epithelial breast cells (Gorlewska, et al. Proc. Am. Assoc. Cancer Res. 43, 1018-1019). Therefore, we sought to determine if 4-ABP, a recognized human urinary bladder carcinogen, is present in commercial hair dyes. 4-ABP was isolated from dyes by solvent extraction with hexane, followed by silica gel chromatography, either with or without chemical treatment of the extract with Zinc/HCl, and a final purification with a mixed cation exchange reversed-phase resin. The identity of 4-ABP was confirmed by both HPLC with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS) following chemical derivatization with pentafluoropropionic anhydride (PFPA). The levels of 4-ABP ranged from not detectable (<0.29 parts per billion (ppb)) up to 12.8 ppb. The noncarcinogenic isomer 2-aminobiphenyl (2-ABP) was also found at quantities up to 310 ppb. 4-ABP was detected in eight of the 11 hair dyes and found in black, red, and blonde hair dyes but not in brown hair dyes. 1,4-Phenylenediamine (PPD) is a key constituent for color development of many permanent hair dyes. Some batches of chemical research grade PPD were contaminated with 4-ABP (up to 500 ppb) and 2-ABP (up to 70 parts per million) and may be a source of ABP contamination in hair dyes. These analytical data demonstrate that 4-ABP is present in some hair dyes. Studies on dermal absorption and bioavailability of 4-ABP from hair dyes are required to determine if this aromatic amine contributes to the increased risk of bladder cancer reported in frequent users of hair dyes.
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