Obligatory free radical intermediate in the oxidative activation of the carcinogen N-hydroxy-2-acetylaminofluorene

We have demonstrated that the nitroxyl free radical form of the carcinogen N-hydroxy-2-acetylaminofluorene (OH-AAF) is an obligatory intermediate in the cumene hydroperoxide-hematin-induced oxidative activation of this carcinogen into 2-nitrosofluorene and N-acetoxy-2-acetylaminofluorene. Both the rate of N-OH-2-acetylaminofluorene oxidation and the amount of its nitroxyl free radical were experimently observed as a function of reaction time. Rate equations were derived for a model in which the nitroxyl free radical form of OH-AAF was an obligatory intermediate in the reaction. Using this theory it was possible to compute one experimental variable, the rate of OH-AAF oxidation, utilizing the other experimental variable, the amount of nitroxyl free radical present at any time during the reaction. The theory also predicts a linear relationship between the rate of OH-AAF oxidation and the square of the free radical content; and this was found to be true experimentally. The dismutation rate of constant of the nitroxyl free radical of OH-AAF was found to be 2.7 · 10⁵ M^?1 · s^?1.     

Quantitative analysis of the ring-hydroxylated metabolites of 2-fluorenylacetamide by gas-liquid chromatography

An analytical procedure is described for quantitative estimation of the C₅- and C₇-hydroxylated metabolites of the carcinogenic amide, 2-fluorenylacetamide. After incubation of 2-fluorenylacetamide in the presence of rat or hamster liver microsomes and a NADPH-generating system, these metabolites were extracted and derivatized with heptafluorobutyrylimidazole. Subsequent analysis by gas-liquid chromatography with electron-capture detection was sensitive enough for determination of the kinetic parameters of these hydroxylation reactions. The effects of pretreatment with 3-methylcholanthrene or 2,3-benzanthracene on these parameters were also studied.     

Arylamidation and arylation by the carcinogen N-2-fluorenylacetamide: A sensitive and rapid radiochemical assay

N-acetoxy-N-arylacetamides, which are generally considered as an ultimate carcinogenic form of the corresponding N-arylacetamides, react with the cellular macromolecules (nucleic acids, proteins, etc.) to give two types of adducts: (I) arylamidation and (II) arylation addition products. In this paper, we present a radiochemical determination of the amount of N-2-fluorenylacetamide bound to DNA via arylamidation or arylation, respectively. This assay is based upon the difference of stability under weak alkali hydrolysis conditions (0.1 N NaOH, 75°C, 2 h) of the specifically ¹⁴C-labeled N-acetyl group of the N-2-fluorenylacetamide residue linked to the macromolecule either via arylamidation or arylation. Native DNA which has been reacted with N-acetoxy-N-2-[¹⁴C]acetylaminofluorene exhibits 16% of the fluorene adducts linked to the bases via arylation. On the other hand, denatured DNA reacts with the fluorene derivative to give almost only arylamidation addition products.     

Procedures for the quantitative analysis of microbial N-dealkylation systems by gas-liquid chromatography

Gas-liquid chromatography methpods were developed for the determination of transformation products and unaltered substrate in mirobial N-dealkylation studies with drug molecules. the microorganism used was the fungus Cunninghemella echinulata, and the drugs employed included morphine alkaloids, various N-substituted 6,7-benzomorphans, and diazepam, a member of the benzodiazepine group of drugs. Assays based on the GLC system utilizing a non-polar SE-30 stationary phase and employing a derivatization step prior to chromatography were devised. The development of these methods facilitated quantitative studies on microbial N-dealkylation and the relationship between substrate structure and microbial transformation activity.     

A novel oxidation of the carcinogen N-hydroxy-N-2-fluorenylacetamide catalyzed by peroxidase/H2O2/Br-

N-Hydroxy-N-2-fluorenylacetamide, a proximate carcinogenic metabolite of N-2-fluorenylacetamide, is oxidized largely to 2-nitrosofluorene by lactoperoxidase or extract of peroxidative activity of rat uterus in an H2O2- and Br- -dependent reaction. Evidence is presented that the oxidizing species includes OBr- (HOBr). This novel oxidation may be involved in carcinogenesis by N-arylhydroxamic acids.     

N-hydroxy-2-fluorenylacetamide, an active intermediate of the mammary carcinogen N-hydroxy-2-fluorenylbenzenesulfonamide.

The mechanism of mammary carcinogenesis by N-hydroxy-2-FBS, a highly potent mammary carcinogen for the female rat by ip administration, has been investigated. Previous work in vivo indicating hydrolytic cleavage of the nitrogen-sulfur bond has been confirmed with the use of sonicates of mammary gland. One of the products of the hydrolysis was N-hydroxy-2-FA identified by its conversion to 2-FA. Since carcinogenicity tests by local application showed that N-hydroxy-2-FA was not carcinogenic for the mammary gland, desulfonylation of N-hydroxy-2-FBS by mammary gland does not account for mammary carcinogenesis. N-Hydroxy-2-FBS applied directly to the mammary gland was not carcinogenic and 2-nitrosofluorene, the product of the spontaneous decomposition of N-hydroxy-2-FBS, exhibited only weak carcinogenicity upon local application. In contrast, N-hydroxy-2-FAA, a urinary metabolite of N-hydroxy-2-FBS, was highly carcinogenic by local application and very likely mediates the action of N-hydroxy-2-FBS. A metabolic pathway for the conversion of N-hydroxy-2-FBS to N-hydroxy-2-FAA is presented. This pathway involves the intermediate formation, by mammary gland or liver, of N-hydroxy-2-FA. The site of the subsequent acetylation of the hydroxylamine is unknown at present although the mammary gland appears to be excluded.     

Discovery of novel N-hydroxy-2-arylisoindoline-4-carboxamides as potent and selective inhibitors of HDAC11

N-Hydroxy-2-arylisoindoline-4-carboxamides are potent and selective inhibitors of HDAC11. The discovery, synthesis, and structure activity relationships of this novel series of inhibitors are reported. An advanced analog (FT895) displays promising cellular activity and pharmacokinetic properties that make it a useful tool to study the biology of HDAC11 and its potential use as a therapeutic target for oncology and inflammation indications.     

A novel assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.     

Mutations to azaguanine resistance induced in cultured diploid human fibroblasts by the carcinogen, N-acetoxy-2-Acetylaminofluorene

The ability of the carcinogen, N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), to induce mutations to azaguanine resistance in diploid human cells was quantitatively investigated and shown to be dose-dependent. The 8-azaguanine (AG) resistance was shown to be heritable in the absence of mutagen or selective agent and the cells of the mutant clones were shown to retain normal sensitivity to N-AcO-AAF.     

Separation of triglycerides by gas-liquid chromatography

The parameters affecting the separation and quantification of triglycerides by gas-liquid chromatography have been investigated with the use of QF-1 and SE-30 as stationary phases and a flame ionization detector. The isothermal characteristics of a wide variety of triglycerides (carbon number 6 to 60) on both columns show that long retention volume is directly proportional to carbon number and inversely proportional to absolute temperature. Isothermal retention indices of some triglycerides are given, as are column efficiencies (in terms of theoretical plates and ability to separate closely related triglycerides). When various rates of programmed temperature rise are used, retention indices have been found to be less useful than absolute or relative elution temperatures. The elution temperatures of triglycerides of carbon number 6 to 54 have been determined relative to that of trilaurin. Under optimal separation conditions weight and molar correction factors can be obtained. Triolein and tristearin have been partially separated, as have certain triglycerides that have the same carbon number but widely different fatty acids. The natural triglycerides of human milk fat have been separated.