Aminofluorenes formation

The metabolic formation of N-sulfonyloxy-N-acetyl-2-aminofluorene (N-sulfonyloxy-AAF) and its observed electrophilic reactivity, provided the first evidence for the importance of enzymatic conjugation reactions in chemical carcinogenesis (23,24). This reaction was shown to be catalyzed by PAPS-dependent sulfotrans-ferases that are located predominantly in liver cytosol and has been subsequently demonstrated for N-hydroxy arylamide metabolites of several other carcinogens, including N-acetyl-4-aminobiphenyl (AABP), benzidine, N-acetyl-2-aminophenanthrene and phenacetin. 

C. C. Lai, E. C. Miller, A. Liem, The Essential Role of Microsomal Deacetylase Activity in the Metabolic Activation, DNA-(Deoxyguanosin-8-yl)-2-aminofluorene Adduct Formation and Initiation of Liver Tumors by A-Hydroxy-2-acetylaminofluorene in the Livers of Infant Male B6C3Fj Mice , Carcinogenesis 1988, 9, 1295-1302. 

Prostaglandin synthetase, peroxidase or lipid peroxidation have been shown to oxidise arylamine xenobiotics to reactive species that bind extensively to DNA. The binding could be an initial event in the toxic or carcinogenic process. Evidence is presented that cation radicals are involved in the formation of the various oxidation products and DNA adduct formation with the carcinogen aminofluorene. Furthermore methylaminoazobenzene (butter yellow) was found to form the same major GSH adduct as is formed in vivo. 

TABLE II. DNA OR POLYRIBONUCLEOTIDE ADDUCT FORMATION WITH AMINOFLUORENE FOLLOWING OXIDATION BY PEROXIDES... 

The addition of H2O2 to the mixtures containing 2-AF and peroxidase at pH 6.5 resulted in the rapid formation of a blue colour with an absorption maxima at 385 and 615 nm which reached a maximum in about 30 sec. The intensity of this colour was directly proportional to the enzyme and aminofluorene concentrations. One equivalent of H2O2 with respect to aminofluorene was required. The blue colour faded after this and became light brown after 5 mins. The blue colour disappeared upon extraction with ethyl acetate. The yellow extract was concentrated and applied to a TLC plate. The Rf of the various products is shown in Table III. Azofluorene was readily identified by its absorption spectra and mass spectrometry. The UV-visible absorption spectra had maxima in ethanol at 278 and 380 nm with shoulders at 362 and 396 nm. The mass spectrum showed a molecular ion at m/e 358.1463... 

Azofluorene formation was decreased if the reacting mixture contained DNA. Two other products decreased by the presence of DNA was a band (Rf 0.31) II which turned brown-green within several hours of running the plate and a band (Rf 0.48) I which turned blue-green after running the plate. Ascorbate was needed to be added to the reaction mixture before extraction to observe band I. This suggests that I unlike II is fully oxidised in the reaction mixture. Oxidation of both products by H202 and peroxidase resulted in the rapid formation of the same transient blue intermediate as was observed with aminofluorene. Product I however bound much more rapidly to DNA than Product II and unlike Product II could not be removed from the DNA by extraction with 1% sodium dodecyl sulfate. Product II showed... 

We previously compared the properties of the reactive species involved in DNA binding by N-OH-aminofluorene, N-acetoxy-acetyl-aminofluorene and the aminofluorene products formed by a peroxidase catalyzed oxidation (28). The marked differences in reactivity suggested that different species were involved and that nitrenium ions were not responsible for the peroxidase catalyzed activation. The identity of the products formed following a peroxidase catalyzed oxidation is still not clear. Other investigators have shown the formation of nitrofluorene and azofluorene but most of the... 

Carbenoid generation of nitrogen ylides represents a useful alternative to the widely employed base-promoted methodology.49 The reaction of aliphatic diazo compounds with tertiary amines was first investigated by Bamford and Stevens in 1952.50 The formation of a-benzyl-a-dimethyl-aminofluorene (99) from the reaction of diazofluorene (97) with ben-zyldimethylamine is consistent with a mechanism involving the generation of ammonium ylide 98 which then undergoes a [l,2]-benzyl shift. 

N-hydroxylation of aromatic amides, which occurs to a minor extent, is of some toxicological intcre.st. since this biotrunsfomiation pathway miiy lead to the formation of chemically reactive intermediates. Several examples of cytotoxicity or carcinogenicity associated with metabolic N-hydroxylation of the parent aromatic amide have been reported. For example, the well-known hepatocareiniigcnic 2-acctyl-aminofluorene (AAF) undergoes an N-hydmxylation reaction catalyzed by cytochrome P-450 to form the cnrre.sp(ind-ing N-hydroxy metabolite (also called a hydroxamic... 

Acetylaminofluorene is not carcinogenic 2-acetylaminofluorene is carcinogenic. 2-Acetylami-nofluorene can be metabolized to form N-hydroxy-acetylaminofluorene and 2-aminofluorene, which may covalently bind to the DNA and macromolecules. Ring hydroxylation, however, leads to the formation and excretion of water-soluble conjugates (e.g., glucuronides) of the respective hydroxylated metabolites and detoxification. 

Tang, M.S., Bohr, V.A., Zhang, X.S., Pierce, J., and Hanawalt, P.C. (1989) Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using... 

Frederick CB, Mays JB, Ziegler DM, Guengerich FP, Kadlubar FF. Cytochrome P450 and flavin-containing monooxygenase-catalyzed formation of the carcinogen N-hydroxy-2-aminofluorene, and its covalent binding to nuclear DNA. Cancer Res 1982 42 2671-2677. 

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