Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx300103jTienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted proteins (i.e. Western blot vs. C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from human and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future

The end product of transglutaminase crosslinking: simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] and lysine by HPLC-MS3.

International audienceTransglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry has been developed for the simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] isodipeptide and lysine on an ion trap mass spectrometer. Highly specific detection has been achieved in MS3 mode. The method includes a derivatization step consisting of butylation of carboxylic groups and acetylation of amide groups, a liquid-liquid extraction, and a 19-min separation on a 100x2.1-mm Beta-basic C18 column with an acetonitrile gradient elution. 13C6-(15)N2 isotopes of the isodipeptide and the lysine serve as internal standards. The assay was linear in the range of 50 pmol/ml to 75 nmol/ml for the isodipeptide and the range of 10 nmol/ml to 3.5 micromol/ml for the lysine, with correlation coefficients greater than 0.99 for both ions. Intra- and inter-day coefficients of variation ranged from 3.5 to 15.9%. The method was successfully applied to human biological samples known to be crosslinked by transglutaminase such as cornified envelopes of epidermis, fibrin, and normal and Huntington disease brain

Unusual Regioselectivity and Active Site Topology of Human Cytochrome P450 2J2

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Étude radiolytique de réactions radicalaires du sulfarlem

Le Sulfarlem est un composé hétérocyclique soufré qui présente des propriétés antioxydantes. L’étude quantitative des mécanismes d’échange monoélectronique impliqués dans cette action a été effectuée par la méthode de la radiolyse continue en solution éthanolique. Elle a permis de montrer que le Sulfarlem ne semble pas réagir avec les radicaux peroxyle ni les ions superoxyde. En revanche, il est un capteur très efficace des radicaux libres non oxygénés de type R•

Role of Arginine 117 in Substrate Recognition by Human Cytochrome P450 2J2

The influence of Arginine 117 of human cytochrome P450 2J2 in the recognition of ebastine and a series of terfenadone derivatives was studied by site-directed mutagenesis. R117K, R117E, and R117L mutants were produced, and the behavior of these mutants in the hydroxylation of ebastine and terfenadone derivatives was compared to that of wild-type CYP2J2. The data clearly showed the importance of the formation of a hydrogen bond between R117 and the keto group of these substrates. The data were interpreted on the basis of 3D homology models of the mutants and of dynamic docking of the substrates in their active site. These modeling studies also suggested the existence of a R117-E222 salt bridge between helices B’ and F that would be important for maintaining the overall folding of CYP2J2

Effect of the watering method on the health of pigs

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Carcinogenicity of benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides on mouse skin.

Benzo[a]pyrene and three arene oxides of benzo[a]pyrene (benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides) have been tested for carcinogenicity in mice by topical application of each compound (0.1 or 0.4 mumol) once every 2 weeks for 60 weeks. At the high dose, benzo[a]pyrene and the 7,8-oxide were highly carcinogenic, whereas the 4,5-oxide (K-region oxide) was weakly active and the 9,10-oxide was inactive. At the low dose, only benzo[a]pyrene was highly carcinogenic. The carcinogenic activities of the three arene oxides of benzo[a]pyrene were not correlated with their stabilities or mutagenic activities