Mechanism-Based Inactivation of Cytochrome P450s 1A2 and 3A4 by

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Mechanism-Based Inactivation of Cytochrome P450s 1A2 and 3A4 by Dihydralazine in Human Liver Microsomes Yasuhiro Masubuchi and Toshiharu Horie* Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Received July 13, 1999

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (antiLM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6β-hydroxylase activity (P450 3A4), but not in diclofenac 4′-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-firstorder kinetics and was saturable with increasing dihydralazine concentrations. Similar timedependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Introduction Antimicrosome autoantibodies commonly appear in the sera of the patients with drug-induced hepatitis. The formation of reactive metabolites of the drugs has been proposed as an initial step in the disease. The step may be followed by covalent binding of the metabolites to the protein generating the reactive metabolite and/or other proteins, which then behave as neoantigens and trigger an abnormal immunological response, leading to the disease. Cytochrome P450 has been proposed as one of the targets of the reactive metabolites in drug-induced hepatitis, because the autoantibodies were found to recognize P450 enzymes (1-4). Dihydralazine, an antihypertensive drug, is known to induce immunoallergic hepatitis on rare occasions (5, 6). The autoantibodies named anti-liver microsomes (antiLM)1 found in the serum of the patients have been reported to be directed against P450 1A2 (3). It is thus suggested that a reactive metabolite of dihydralazine, which might be generated by P450 1A2, covalently binds * To whom correspondence should be addressed: Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. Phone: 8143-290-2934. Fax: 81-43-290-3021. E-mail: [email protected]. 1 Abbreviations: anti-LM, anti-liver microsomes; GSH, reduced glutathione; G-6-P, glucose 6-phosphate; G-6-PDH, glucose-6-phosphate dehydrogenase.

to the P450 itself and triggers an immunological response as a neoantigen. In practice, a study with 14C-labeled dihydralazine demonstrated that dihydralazine was activated by rat P450 1A2 into a chemically reactive metabolite that covalently binds to liver microsomal protein, and suggested that the reactive metabolite bound to P450 1A2 in rat and human liver microsomes (7). However, the question of whether P450 1A2 is the sole target of the dihydralazine metabolite remains in doubt, since we recently observed in rat liver microsomes the fact that dihydralazine metabolism caused nonselective inactivation of P450 enzymes, i.e., not only P450 1A2 but also P450s 2C11 and 3A (8). We investigated inactivation of human P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and target of dihydralazine.

Experimental Procedures Chemicals. Dihydralazine dihydrochloride was purchased from Aldrich (Milwaukee, WI). Phenacetin, 4-acetamidophenol, and testosterone were from Sigma Chemical Co. (St. Louis, MO). Diclofenac sodium and reduced glutathione (GSH) were from the Wako Pure Chemical Ind. (Osaka, Japan). 6β-Hydroxytestosterone was from Steraloids Inc. (Wilton, NH). 4′-Hydroxydiclofenac was from Gentest Co. (Woburn, MA). Glucose 6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH),

10.1021/tx9901276 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999

Dihydralazine Inactivates P450s 1A2 and 3A4

Figure 1. Time- and concentration-dependent decreases in phenacetin O-deethylase activity in human liver microsomes during dihydralazine metabolism. The microsomal reaction mixture was preincubated without (O) or with dihydralazine [12.5 (b), 25 (4), 50 (2), and 100 µM (0)] for various periods of time in the presence of NADPH, followed by an assay of phenacetin O-deethylase activity. Results are means ( SE of three determinations. and NADPH were from Oriental Yeast Co., Ltd. (Tokyo, Japan). All other chemicals and solvents were analytical grade. Liver Microsomes and P450 Enzymes. Human liver microsomes (pooled fraction from 12 patients) and microsomal preparations from B-lymphoblastoid cell lines expressing P450s 1A2 and 3A4 were purchased from Gentest Co. Protocols for Preincubation of Liver Microsomes with Hydralazine Derivatives. Pooled human liver microsomes and microsomes from lymphoblastoid cells expressing specific P450s were preincubated with dihydralazine in the presence of NADPH, to determine the effects of the metabolites on microsomal monooxygenase activities. A 1 mL incubation mixture contained 0.25 mg of liver microsomal protein, 10 mM G-6-P, 2 units of G-6-PDH, 10 mM MgCl2, 0.1 mM EDTA, and various concentrations of dihydralazine in 0.15 M potassium phosphate buffer (pH 7.4). Tris-HCl buffer (0.15 M, pH 7.4) was also used for the mixture instead of the phosphate buffer to determine the effect on diclofenac 4′-hydroxylase activity. After temperature equilibration (37 °C, 5 min), preincubation of microsomes with dihydralazine was started by adding NADPH (final concentration of 0.5 mM) and performed for various periods of time. The subsequent incubation of the microsomes for the assay of enzymatic activities was started by addition of each test substrate. In the control experiments, the same reaction mixture as described above, including the corresponding concentrations of dihydralazine, was not preincubated prior to the incubation for the assay of enzyme activities, or the mixture without dihydralazine was preincubated for the corresponding periods of time. Assay of Enzymatic Activities. Phenacetin O-deethylase (9), testosterone 6β-hydroxylase (10), and diclofenac 4′-hydroxylase (11) activities were determined according to the HPLC method previously described. Substrate concentrations were 25 µM phenacetin, 200 µM testosterone, and 20 µM diclofenac. All of the assays were performed under linear conditions of metabolite formation with regard to incubation time and protein concentration. Data Analysis. Pseudo-first-order kinetic constants for the enzyme inactivation (k) were calculated from the initial slopes of the linear regression lines of semilogarithmic plots of the remaining activity against the preincubation time. The reciprocal of k thus obtained was plotted against the reciprocal of the dihydralazine concentration, and then a maximal rate constant for inactivation (kinact) and the concentration at which the rate constant for inactivation is half-maximal (KI) were determined from the intercepts on the ordinate and the abscissa, respectively. Results were represented as means ( SE. Statistical significance was calculated by the Student’s t test.

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Figure 2. Time- and concentration-dependent decreases in testosterone 6β-hydroxylase activity in human liver microsomes during dihydralazine metabolism. The microsomal reaction mixture was preincubated in the same manner as described in the legend of Figure 1, followed by an assay of testosterone 6βhydroxylase activity. Results are means ( SE of three determinations.

Figure 3. Lack of an effect of dihydralazine metabolism on diclofenac 4′-hydroxylase activity in human liver microsomes. The microsomal reaction mixture was preincubated without (O) or with dihydralazine [50 µM (b)] for various periods of time in the presence of NADPH, followed by an assay of diclofenac 4′hydroxylase activity. Results are means ( SE of three determinations.

Results The effects of addition of dihydralazine and of preincubation of microsomes with dihydralazine in the presence of NADPH were tested by using human liver microsomes, which are commercially available. Figures 1-3 show the effects on phenacetin O-deethylase, testosterone 6β-hydroxylase, and diclofenac 4′-hydroxylase activities, which are mainly mediated by P450s 1A2, 3A4, and 2C9, respectively, in human liver microsomes. Addition of dihydralazine slightly diminished phenacetin O-deethylase activity, whereas the decrease in the activity was intensified by the preincubation of microsomes with dihydralazine and NADPH in a time-dependent manner (Figure 1). Dihydralazine also caused a decrease in testosterone 6β-hydroxylase activity, and the preincubation also caused a time-dependent loss of the enzyme activity (Figure 2). On the other hand, none of the effect of addition of dihydralazine (50 µM) was observed on diclofenac 4′-hydroxylase activity during any time period of the preincubation (Figure 3). These results indicate that P450s 1A2 and 3A4 but not 2C9 were inactivated during the metabolism of dihydralazine. Phenacetin O-deethylase and testosterone 6β-hydroxylase activities decreased exponentially with respect to the

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Figure 4. Double-reciprocal plots of the first-order rate constant for inactivation of phenacetin O-deethylase activity vs dihydralazine concentration. The microsomal reaction mixture was preincubated with various concentrations of dihydralazine in the presence of NADPH, followed by an assay of phenacetin O-deethylase activity. Pseudo-first-order kinetic constants for the enzyme inactivation (k) were calculated from the initial slopes of the linear regression lines of semilogarithmic plots of the remaining activity vs the preincubation time, which were shown in Figure 1. The reciprocal of k thus obtained was plotted against the reciprocal of the dihydralazine concentration. kinact and KI were calculated to be 0.0260 ( 0.0018 min-1 and 41.9 ( 8.2 µM, respectively. Results are means ( SE of three determinations.

Figure 5. Double-reciprocal plots of the first-order rate constant for inactivation of testosterone 6β-hydroxylase activity vs dihydralazine concentration. Pseudo-first-order kinetic constants for the enzyme inactivation (k) were obtained from Figure 2 in the same manner as described in the legend of Figure 4. kinact and KI were calculated to be 0.0495 ( 0.0076 min-1 and 35.0 ( 12.3 µM, respectively. Results are means ( SE of three determinations.

preincubation time, and the first-order kinetic constants for the enzyme inactivation (k) obtained at various dihydralazine concentrations indicated saturation of the inactivation with increasing dihydralazine concentrations. Double-reciprocal plots of the first-order rate constant for inactivation of phenacetin O-deethylase (Figure 4) and testosterone 6β-hydroxylase (Figure 5) as a function of dihydralazine concentration provided the kinetic parameters for the enzyme inactivation. The maximal rate constant for inactivation of phenacetin O-deethylase (kinact) and the concentration at which the rate constant for inactivation is half-maximal (KI) were calculated to be 0.0260 ( 0.0018 min-1 and 41.9 ( 8.2 µM, respectively (mean ( SE, n ) 3). The corresponding kinetic parameters for testosterone 6β-hydroxylase were calculated to be 0.0495 ( 0.0076 min-1 and 35.0 ( 12.3 µM, respectively.

Masubuchi and Horie

Figure 6. Effect of GSH on the decrease in phenacetin O-deethylase activity in human liver microsomes during dihydralazine metabolism. The microsomal reaction mixture was preincubated with NADPH in the absence or presence of dihydralazine (50 µM) and GSH (5 mM) for 20 min, followed by an assay of phenacetin O-deethylase activity. Results are means ( SE of three determinations. One asterisk indicates a result significantly different from that for the dihydralazine (-) group (p < 0.05). Two asterisks indicate a result significantly different from that for the dihydralazine (-) group (p < 0.01).

Figure 7. Effect of GSH on the decrease in testosterone 6βhydroxylase activity in human liver microsomes during dihydralazine metabolism. The microsomal reaction mixture was preincubated with NADPH in the absence or presence of dihydralazine (50 µM) and GSH (5 mM) for 20 min, followed by an assay of testosterone 6β-hydroxylase activity. Results are means ( SE of three determinations. Two asterisks indicate a result significantly different from that for the dihydralazine (-) group (p < 0.01).

Human liver microsomes were preincubated with dihydralazine and NADPH in the presence or absence of GSH to determine its protective effect against the inhibition of phenacetin O-deethylase activity by dihydralazine metabolism. The decrease in the activity by the preincubation of microsomes was observed both without and with GSH at the concentration of 5 mM (Figure 6). A similar lack of a protective effect of GSH was observed for the inhibition of testosterone 6β-hydroxylase activity (Figure 7). The effects of dihydralazine were also tested using microsomes from B-lymphoblastoid cells expressing P450 enzymes instead of human liver microsomes. Figures 8 and 9 show the effects on P450 1A2-mediated phenacetin O-deethylase activity and P450 3A4-mediated testosterone 6β-hydroxylase activity, respectively, at a dihydralazine concentration of 50 µM. Results similar to those for human liver microsomes were obtained. Namely, phenacetin O-deethylase activity was decreased exponentially by the preincubation of the microsomes with dihydrala-

Dihydralazine Inactivates P450s 1A2 and 3A4

Figure 8. Time-dependent decreases in phenacetin O-deethylase activity of recombinant P450s 1A2 during dihydralazine metabolism. The reaction mixture of microsomes from human B-lymphoblastoid cells expressing P450 1A2 was preincubated without (O) or with dihydralazine [50 µM (b)] for various periods of time in the presence of NADPH, followed by an assay of phenacetin O-deethylase activity. Results are means ( SE of three determinations.

Figure 9. Time-dependent decreases in testosterone 6βhydroxylase activity of recombinant P450s 3A4 during dihydralazine metabolism. The reaction mixture of microsomes from human B-lymphoblastoid cells expressing P450 3A4 was preincubated without (O) or with dihydralazine [50 µM (b)] for various periods of time in the presence of NADPH, followed by an assay of testosterone 6β-hydroxylase activity. Results are means ( SE of three determinations.

zine in the presence of NADPH (Figure 8). The preincubation of microsomes also caused a decrease in testosterone 6β-hydroxylase activity in a time-dependent manner (Figure 9). The first-order kinetic constants for the inactivation of P450s 1A2 and 3A4 obtained at the dihydralazine concentration of 50 µM were 0.0192 ( 0.0023 and 0.0471 ( 0.0006 min-1, respectively.

Discussion Since the autoantibodies in the serum of patients with dihydralazine hepatitis recognized P450 1A2, it is suggested that a reactive metabolite of dihydralazine, which might be generated by P450 1A2, covalently binds to the P450 itself (3). In vitro data supported the hypothesis that the metabolite bound covalently to P450 1A2 (7), whereas our recent observation with rat liver microsomes suggested that P450 isozymes other than P450 1A2 also were targets of the dihydralazine metabolite (8). Thus, we have attempted to determine whether human P450 1A2 is the specific target protein of the reactive metabolite. In the situation where the radiolabeled drug or

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specific antibody against the covalent adduct is not available, the fact that metabolism-dependent inactivation of the enzyme is assessed by alteration of its catalytic activity is valuable. Indeed, tienilic acid, which induces immunoallergic hepatitis and is proposed to bind covalently to P450 2C9, has been shown to be a mechanismbased inactivator of P450 2C9 (12). Thus, we investigated the inactivation of human P450 enzymes during the metabolism of dihydralazine. Preincubation of microsomes from human liver with dihydralazine in the presence of NADPH resulted in a marked decrease in the phenacetin O-deethylase activity (Figure 1), demonstrating oxidative metabolism of dihydralazine caused inactivation of P450 1A2. A similar metabolism-dependent decrease in the activity was observed for testosterone 6β-hydroxylase activity (Figure 2) but not for diclofenac 4′-hydroxylase activity (Figure 3). These data indicated that P450 3A4 as well as P450 1A2 was a target of a reactive metabolite of dihydralazine in human liver microsomes. To determine whether the decrease in the activities of these two P450 enzymes occurs via the mechanism-based inactivation process, kinetic analysis of the inactivation was performed with human liver microsomes. The pseudofirst-order kinetics for the time-dependent inactivation and saturability of inactivation with increasing dihydralazine concentrations observed in both phenacetin O-deethylase and testosterone 6β-hydroxylase activities (Figures 4 and 5) suggested the mechanism-based inactivation of P450s 1A2 and 3A4. In addition, the inactivation constants showed that inactivation of P450 3A4 was more pronounced than that of P450 1A2. On the other hand, GSH did not prevent dihydralazine-induced inactivation of the P450 enzymes (Figures 6 and 7), supporting the postulated process, because the lack of the prevention by GSH is one of the characteristics of the mechanism-based inactivation. Finally, a single P450 enzyme instead of liver microsomes was used for evaluation of effects of dihydralazine metabolism. As clearly shown in Figures 8 and 9, results similar to those for liver microsomes were obtained from preincubation of P450s 1A2 and 3A4 expressed in lymphoblastoid cells with dihydralazine. Therefore, inactivation of P450 1A2 was shown to be attributed to a reactive metabolite formed by P450 1A2 itself, and inactivation of P450 3A4 was attributed to that by P450 3A4. Namely, dihydralazine is demonstrated to be a mechanism-based inactivator of both P450 1A2 and 3A4. The data presented here support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM autoantibodies in dihydralazine hepatitis, whereas the data also suggest that the appearance of autoantibodies specifically recognizing P450 1A2 is not attributed to selective covalent binding of its reactive metabolites to P450 1A2, since P450 3A4 was also shown to be a target of dihydralazine metabolites. The fact that an autoantibody against P450 3A4 did not appear in the patients indicates that P450 3A4 could not be obtained for the antigenicity by the covalent binding of dihydralazine metabolites; that is, the covalent binding to P450 3A4 might not result in activation of either B- or T-cells by T-helper cells, which could recognize only modified self-peptides (13, 14). Another explanation is that even if the epitope was formed by the covalent binding, it might lose immunogenicity during degradation of the P450 protein into

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small peptides before recognition by the immune system. However, the latter situation is not possible because the short linear epitope was suggested to be that of P450 3A enzymes by the recent epitope mapping analysis with anti-P450 3A autoantibody in the sera of patients of anticonvulsant-induced hypersensitivity reactions (15), whereas the epitope of P450 1A2 was suggested to be conformational (16). It is accepted that the specificity of the covalent binding to a P450 isoform, which probably resulted from selective metabolic activation by the P450, causes the appearance of autoantibody against the specific P450 (2-4, 17). The results presented here provide negative suggestion for the speculation. To clarify the mechanism of appearance of autoantibody to specific P450, it seems to be valid to consider possibilities other than the catalytic activity, essentially, processes after the covalent binding and structural characteristics of P450 enzymes in forming epitope preferably. Further studies are required for elucidation of these points, and for defining the mechanism of appearance of autoantibodies against P450 enzyme in hypersensitivity reactions, as well as for elucidation of whether these autoantibodies are just side products of the pathogenic process or are directly involved in liver cell injury.

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Acknowledgment. We thank Mr. J. Ose for his technical assistance.

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