Human Liver Mephenytoin 4 -Hydroxylase Cytochrome P-450 Proteins and Genes

Brian, William R.; Gedt, Cecile; Bellew, Teresa M.; Srivastava, Pramod K.; Bork, Richard W.; Umbenhauer, Diane R.; Lloyd, R. Stephen; Guengerich, F. Peter

doi:10.3109/03602538909103552

Cited by 6 publications

(3 citation statements)

References 21 publications

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“…Saccharomyces cerevisiae strain D12 [a, leu - , (cir + )] was used for expression. Details of protocols for construction of vectors were presented elsewhere ( − ).…”

Section: Methodsmentioning

confidence: 99%

Opposite Behaviors of Reactive Metabolites of Tienilic Acid and Its Isomer toward Liver Proteins: Use of Specific Anti-Tienilic Acid−Protein Adduct Antibodies and the Possible Relationship with Different Hepatotoxic Effects of the Two Compounds

Bonierbale

Valadon

Pons

et al. 1999

Chem. Res. Toxicol.

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Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA. Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA). These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins. This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9. TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450 responsible for its hepatic activation in humans. On the contrary, TAI showed a nonspecific alkylation pattern, targeting many proteins upon metabolic activation. Nevertheless, this nonspecific covalent binding could be completely shifted to a thiol trapping agent like GSH. The difference in alkylation patterns for these two compounds is discussed with regard to their distinct toxicities. A relationship between the specific covalent binding of P450 2C9 by TA and the appearance of the highly specific anti-LKM2 autoantibodies (known to specifically recognize P450 2C9) in patients affected with TA-induced hepatitis is strongly suggested.

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“…Saccharomyces cerevisiae strain D12 [a, leu - , (cir + )] was used for expression. Details of protocols for construction of vectors were presented elsewhere ( − ).…”

Section: Methodsmentioning

confidence: 99%

Opposite Behaviors of Reactive Metabolites of Tienilic Acid and Its Isomer toward Liver Proteins: Use of Specific Anti-Tienilic Acid−Protein Adduct Antibodies and the Possible Relationship with Different Hepatotoxic Effects of the Two Compounds

Bonierbale

Valadon

Pons

et al. 1999

Chem. Res. Toxicol.

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“…Gly417 for 2C9 and 2C10, respectively). Their cDNAs, which vary in only two bases in the coding region (Umbenhauer et al, 1987;Ged et al, 1988;Brian et al, 1989a) have been classed at present as separate genes on the basis of considerable differences in the 3'-noncoding region but could be considered as allelic variants of the same protein. Whether these minor differences in the primary structure of P450 2C9 and P450 2C10 affect.…”

Section: Discussionmentioning

confidence: 99%

“…The isolation and sequencing of the human cDNA MP-4 (2C9) and MP-8 (2C10) clones has been previously reported (Umbenhauer et al, 1987;Ged et al, 1988;Brian et al, 1989a). These DNA sequences were inserted in the pAAH5 vector (based on an ADHl promoter and terminator) that contains the leu2 gene.…”

Section: Yeast Transformation Cell Culture and Preparation Of Yeastmentioning

confidence: 99%

Human‐liver cytochromes P‐450 expressed in yeast as tools for reactive‐metabolite formation studies

Garcia

Dansette

Valadon

et al. 1993

European Journal of Biochemistry

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Human liver cytochromes P-450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of tienilic acid (2-aryloxo-thiophene) and its isomer (3-aroylthiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5-hydroxylation of tienilic acid by NADPH and 0, (K, = 6 pM, V,, = 2.5 turnover/min), (b) the activation of tienilic acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the tienilic acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1Al and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of tienilic acid (and its isomer) by yeast-expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous K, values (around 10 pM) for tienilic acid 5-hydroxylation, (b) a strong inhibition of tienilic acid oxidation by human sera containing anti-(liver kidney microsomes type 2) (anti-LKM,) antibodies, and (c) almost identical relative ratios of tienilic acid metabolic activation/5-hydroxylation and of tienilic acid activatiodthe activation of its isomer with both systems. Rates of oxidation of tienilic acid (and its isomer) by yeast microsomes are 6-8-fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of tienilic acid in human liver, but also indicate that the 5-hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites.Very few data are presently available about the oxidative metabolism of thiophene compounds (Rance, 1989) and the possible toxicological consequences of this metabolism. Several drugs containing a thiophene ring substituted at position 2 by an aroyl group were reported to be hydroxylated at position 5 of the thiophene ring (Rance, 1989). This is the case for tienilic acid (TA), an uricosuric diuretic drug, in rat and man . In fact, during the hydroxylation of TA which is catalyzed in human liver by a cytochrome P-450 (P450) of the 2C subfamily, electrophilic reactive metabolites are formed and covalently bind to liver proteins . TA

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