Article pubs.acs.org/crt
Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin Xiaoli Meng,† James L. Maggs,† Toru Usui,† Paul Whitaker,‡ Neil S. French,† Dean J. Naisbitt,† and B. Kevin Park*,† †
MRC Center for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool L69 3GE, United Kingdom ‡ The Department of Respiratory Medicine, St. James’s Hospital, Leeds LS9 7TF, West Yorkshire, United Kingdom S Supporting Information *
ABSTRACT: Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.
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conjugate10 in complete microsomal incubations. These observations suggest that protein adduction might occur principally by acylation of the side chains of lysine residues. Direct confirmation that INH-protein adducts are formed in cells was achieved recently by Lu et al. using proteomic liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis.13 They identified isonicotinoylated prohibitin 2 and macrophage migration inhibitory factor, acylated at a lysine and proline residue, respectively, in human induced pluripotent stem cell derived hepatocytes. Similar analysis of S9 fractions of livers from INH-treated rats and mice identified D-dopachrome decarboxylase with an IN-lysine residue.14 However, none of these studies have investigated the covalent binding of INH to proteins in the absence of P450, nor have these adducts been detected in human patients exposed to INH therapy. Therefore, the aims of this study were to investigate the nature of INHprotein adducts formed in vitro by using human serum albumin (HSA) as a model protein and to characterize the INH-protein adducts formed in patients taking INH therapy.
INTRODUCTION Isoniazid (INH) remains a first line drug for the treatment of tuberculosis, even though its use is associated with the development of drug-induced liver injury (DILI).1 It has been reported that liver dysfunction occurs in 18−20% of patients receiving INH, but the majority of cases are mild reactions. It has been estimated that overt hepatotoxicity occurs in up to 1% of patients.2−4 The mechanisms underlying INH DILI remain ill-defined; however, there is evidence that the immune system may be involved. For instance, T cells from patients with INH DILI have been shown to proliferate ex vivo in the presence of INH and INH-modified proteins.3 In addition, antibodies against INH-modified proteins have been detected in patients with INH DILI.5 Together, these studies indicate that INHmodified proteins play an important role in the activation of a drug-specific immune response. INH is transformed to form structurally diverse stable metabolites in humans, including acetylhydrazine, isonicotinic acid (INA), and isonicotinic amide, which are potential products or precursors of chemically reactive species.2,6−8 It has been long debated on which reactive intermediates and pathways are involved in the formation of INH-protein adducts and which structural types of adduct are formed. Previous 14Clabeling studies have shown that acetylhydrazine contributed to covalent binding through acetyl radicals.8 Conversely, recent studies by Metushi et al. have proposed that INH could bind to hepatic microsomal proteins through a P450-generated acyl diazene (acyl diimide)9 or diazohydroxide intermediate.10 A reactive metabolite of INH is trapped as an isonicotinic amide of N-α-acetyl-lysine (NAL)10−12 but not as a glutathione © 2014 American Chemical Society
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EXPERIMENTAL PROCEDURES
Chemicals. HSA (97−99% pure) and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO), HLM was prepared from an adult liver sample obtained from a local hospital (Aintree University Hospital NHS Foundation Trust) with informed consent under ethical approval.15 Trypsin was from Promega (Madison, WI), liquid chromatography−mass spectrometry (LC-MS) grade solvents Received: July 12, 2014 Published: November 27, 2014 51
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Table 1. INH-Modified HSA Tryptic Peptides Detected in Patientsa modified lysine residues patient
gender
age
diagnosis
055
F
36
tuberculous lymphadenitis
056
F
60
070
F
63
081
M
81
chemoprophylaxis for antiTNF drugs chemoprophylaxis for antiTNF drugs pulmonary tuberculosis
a
INH dose 300 mg daily 300 mg daily 300 mg daily 300 mg daily
medication
adverse reactions
K137
K190
K525
INH/RIF/ PZA/ETB INH/RIF
cutaneous reactions after 48 h
−
+
+
elevated ALT of 502 U/L, 6 weeks into treatment
−
+
+
INH/RIF
elevated ALT of 347 U/L, 4 weeks into treatment
+
+
+
INH/RIF/ PZA/ETB
elevated ALT of 588 U/L and cutaneous reactions after 4 weeks of treatment
+
+
+
The drug-modified (isonicotinoylated) tryptic peptides of HSA isolated from patients’ plasma were characterized by LC-MS/MS. phosphate buffer, HLM (suspended in phosphate buffer; final protein concentration, 1 mg/mL), or HLM in the presence of NADPH (0.5 mM) were incubated with NAL (1 mM) at 37 °C for 3 h. The control experiment was performed by incubation of INH with HLM and NADPH in the absence of NAL under the same conditions. At the end of the incubation, bovine serum albumin (final concentration, 1 mg/ mL) was added to the mixture in phosphate buffer to remove components and products of the incubation that might cause matrix effects during LC-MS analyses. All samples were spiked with pyronaridine (final concentration, 100 nM) as an internal standard and extracted immediately with cold methanol. The extracts were evaporated to dry residues, reconstituted in 2% acetonitrile/0.1% formic acid and analyzed by LC-MS. Mass Spectrometric Characterization of INH NAL or NADP Conjugates. Samples were delivered into a 4000 QTRAP (AB Sciex, Framingham, MA,) coupled with a 1260 Infinity Quaternary Pump HPLC system (Agilent Technologies, Santa Clara, CA) and Kinetex C18 column (2.6 μm C18, 100 mm × 2.1 mm, Phenomenex, Macclesfield, Cheshire, U.K.). A gradient from 1% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 12 min was applied at a flow rate of 150 μL/min. Mass spectral analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) triggered enhanced product ion scan for quantitative analysis. The ionspray voltage was set to 4.5 kV, and the source temperature was maintained at 450 °C. High purity nitrogen was used for the GAS 1, GAS 2, curtain, and collision gases. The MRM transitions for INH conjugates were as follows: m/z 294 → 106 and m/z 294 → 189 for NAL conjugates and m/z 850.8 → 228 and 850.8 → 136 for NADP conjugates. Relative quantification of INH conjugates was performed by comparing the intensities of MRM peaks normalized against an internal standard (pyronaridine; m/z 518 → 447). Data were analyzed using Analyst software, version 1.5.1 (AB Sciex). Isolation of HSA from Plasma by Affinity Chromatography. HSA was isolated from patients’ plasma by affinity chromatography as described previously17 with slight modifications. An Affinity Removal Column HSA Only (Agilent Technologies) attached to a Vision Workstation (Applied Biosystems) was used to affinity-capture HSA from 50 μL of freshly thawed plasma. The HSA was eluted with 12 mM HCl and precipitated with 9 volumes of ice-cold methanol. The eluted protein was processed for LC-MS/MS analysis as described above. Covalent Binding of INH to HSA in Human Plasma in Vitro. Plasma from healthy volunteers was incubated with INH (freshly dissolved in potassium phosphate buffer) at concentrations of 26.5 μM (3.64 μg/mL, median Cmax plasma concentration in slow acetylators),18 100 μM, or 10 mM at 37 °C. Aliquots of 50 μL were removed after 0.5, 1, 2, 3, 15, and 24 h and processed for LC-MS/MS analysis as described above. Mass Spectrometric Characterization of INH-Modified HSA. Samples were reconstituted in 2% acetonitrile/0.1% formic acid (v/v), and aliquots of 2.4−5 pmol of tryptic digests were delivered into a QTRAP 5500 (AB Sciex) fitted with a NanoSpray II source by automated in-line liquid chromatography (U3000 HPLC System, 5 mm C18 nanoprecolumn and 75 μm × 15 cm C18 PepMap column;
were from Fisher Scientific UK Ltd. (Loughborough, Leicestershire), and all other standard reagents were from Sigma-Aldrich. Patients. Plasma samples were isolated from four patients with hepatic and/or cutaneous adverse drug reactions (ADRs) to INH, and stored immediately at −80 °C. Table 1 summarizes the patients’ demographics and the clinical features of the ADRs. Plasma samples from four patients administered with INH without ADRs were also included in this study. Approval for the study was acquired from the Liverpool and Leeds local research ethics committees; informed written consent was obtained from each patient. Blank plasma was obtained from healthy volunteers who gave informed consent according to a procedure approved by the University of Liverpool Committee of Research Ethics. Concentration-Dependent Modification of HSA by INH in Vitro. INH freshly dissolved in potassium phosphate buffer (10 mM, pH 7.4) was incubated with HSA (0.6 mM, 50 μL) or a serumsupplemented cell culture medium (R9 medium: RPMI 1640 supplemented with 10% pooled heat-inactivated human AB serum, 25 mM HEPES buffer, 2 mM L-glutamine, and 25 μg/mL transferrin) in sealed Eppendorf tubes at 37 °C for 16 h. The molar ratios of INH to protein were 0.1:1, 1:1, 10:1, and 20:1. Protein was precipitated twice with 9 volumes of ice-cold methanol to remove noncovalently bound INH, resuspended in 50 μL of phosphate buffer, and then reduced with 10 mM dithiothreitol (15 min) and alkylated with 55 mM iodoacetamide (15 min) at room temperature. The protein was precipitated with methanol once more and finally dissolved in 100 μL of 50 mM ammonium hydrogen carbonate, and 165 μg (1.25 nmol) of protein was digested with 1.6 μg of trypsin overnight at 37 °C. The samples were processed for LC-MS/MS analysis as described previously.16 Time-Dependent Modification of HSA by INH in Vitro. INH freshly dissolved in potassium phosphate buffer (10 mM, pH 7.4) was incubated with HSA (0.6 mM, 300 μL) at 37 °C (final INH concentration, 6 mM). Aliquots of 50 μL were removed after 0.5, 1, 2, 3, 16, and 24 h and processed for LC-MS/MS analysis as described above. Inhibition of Covalent Binding of INH to HSA. INH (6 mM) was incubated with HSA (0.6 mM, 50 μL) at 37 °C in either phosphate buffer (10 mM, pH 7.4) or phosphate buffer that had been purged with nitrogen for at least 10 min immediately beforehand. Another set of incubations was prepared from buffered solutions of INH (6 mM) and HSA (0.6 mM, 50 μL) spiked with ascorbic acid, GSH, or NAL at 0.01 mM, 0.1 mM, and 1 mM. At the end of the incubation (16 h), protein was precipitated twice with 9 volumes of ice-cold methanol to remove noncovalently bound drugs and processed for LC-MS/MS analysis as described above. Formation of IN-NAL Conjugates in Various Media. Freshly prepared INH solution (10 mM, in either LC-MS grade water, or phosphate buffer, phosphate buffer containing EDTA (10 μM), or phosphate buffer purged with nitrogen for at least 10 min was incubated with NAL (10 mM) at 37 °C for 16 h. The resulting products were spiked with 50 nM pyronaridine as an internal standard and analyzed by LC-MS. Formation of IN-NADP Conjugates in Human Liver Microsomes. Freshly prepared solutions of INH (100 μM or 1 mM) in 52
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Figure 1. LC-MS/MS analysis of an INH-modified albumin peptide identified in incubations with HSA, R9 medium, and plasma. (a) MS/MS spectrum of the INH-modified tryptic peptide 182LDELRDEGK(INA)ASSAK195 with a mass addition of 105 amu; (b) proposed pathway for the generation of the fragment ion of INA-lysine at m/z 189 by loss of CO and NH3. Dionex, Sunnyvale, CA) via a 10 μm inner diameter PicoTip (New Objective, Woburn, MA). A gradient from 2% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 60 min was applied at a flow rate of 300 nL/min. The ion spray potential was set to 2,200−3,500 V, the nebulizer gas to 19, and the interface heater to 150 °C. MRM transitions specific for INH-modified peptides were selected as follows: the m/z values were calculated for all possible peptides containing a lysine residue together with the characteristic fragment ion of m/z 189. MRM transitions were acquired at unit resolution in both the Q1 and Q3 quadrupoles to maximize specificity. They were optimized for collision energy and collision cell exit potential, and the dwell time was 20 ms. MRM survey scans were used to trigger enhanced product ion scans of INH-modified peptides, with Q1 set to unit resolution, dynamic fill selected, and dynamic exclusion for 20 s. Total ion counts were determined from a second aliquot of each sample analyzed by conventional LC-MS and were used to normalize sample loading on the column. Relative quantification of modified peptides was performed by comparing the relative normalized intensity of MRM peaks for each of the modified residues against total ion counts across samples. Data were analyzed using Analyst software (AB Sciex). Statistical Analysis. Data are presented as the means and their standard deviations. Statistical comparisons of experimental groups were made using the paired t tests method (Sigmaplot 12 software, Systat software Inc., San Jose, CA). A value of P < 0.05 was considered to be significant.
of any oxidative enzymes or supplementary redox-active metals. INH modification was evidenced by the MS/MS spectrum shown in Figure 1a.The isonicotinic amide adduct of Lys190 resulted in a doubly charged ion at m/z 812.5, corresponding to a mass increase of 105 amu to the peptide 182LDELRDEGKASSAK195. The peptide sequence was confirmed by a series of b and y product ions, and the site of modification was confirmed by the presence of a9* + 2 (m/z 567.1), b9* + 2 (m/z 581.1), and y6* + 2 (m/z 694.9). A characteristic fragment ion at m/z 189 amu was also observed, corresponding to the INH adducted lysine immonium ion with the loss of an ammonia molecule (Figure 1b). The INH modification appeared to be concentration- and time-dependent (Figure 2a and b). A semiquantitative analysis of modification at each site was performed by determining the peak area for the extracted masses of the modified peptides, followed by normalization of the ion intensity against the internal standard. A concentration- and time-dependent increase in normalized ion counts was observed for the modified K137, K212, and K525 peptides (Figure 2a and b). Furthermore, when INH was incubated with a serumsupplemented cell culture medium, a similar profile of lysine adduction was observed (Figure 2c). Inhibition of the Auto-oxidation of INH in Vitro. In order to further elucidate the auto-oxidation of INH to reactive intermediates, the following experiments were performed. First, INH was incubated with HSA at a molar ratio of 10:1 in phosphate buffer or buffer that had been purged with nitrogen. The yield of the covalent protein adducts was dramatically reduced by the expulsion of dissolved oxygen (Figure 3a). Second, ascorbic acid, GSH, and NAL were coincubated individually with INH and HSA. The formation of the covalent
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RESULTS Characterization of INH-Modified HSA in Vitro. To probe the chemical basis of INH’s covalent binding to HSA, INH was incubated directly with HSA in phosphate buffer (10 mM and pH 7.4). After 16 h, the protein was processed and digested with trypsin as described previously.19 The resulting tryptic peptides were analyzed by LC-MS/MS. Surprisingly, INH-modified peptides were readily detectable in the absence 53
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Figure 3. Characteristics of the spontaneous chemical activation of INH in vitro. (a) Covalent binding of INH to HSA was inhibited by purging oxygen from the solution with nitrogen and by ascorbic acid (ASC) and GSH. (b) Conjugation of INH with NAL was oxygendependent and catalyzed by the trace, redox-active, EDTA chelatable metals present in phosphate buffer (PB). (c) The IN-NAL conjugate was formed in phosphate buffer, in the presence of human liver microsomes (1 mg/mL) without NADPH, or with NADPH (0.5 mM); the control experiment was performed by incubation of INH with HLM and NADPH in the absence of NAL. Data are expressed as the means ± SDs from three different experiments. Statistical comparisons of groups were determined by use of paired t tests; *p < 0.05 was considered to be significant.
Figure 2. LC-MS/MS analysis of INH-modified albumin peptides identified via in vitro incubation with HSA and R9 medium. Modification of some of the lysine residues by INH in phosphate buffer was (a) concentration dependent and (b) time dependent (INH/HSA molar ration at 10:1). (c) Similar lysine adduct profiles were observed when HSA and R9 medium were incubated with INH. (R9 medium: RPMI 1640 supplemented with 10% pooled heat inactivated human AB serum, 25 mM HEPES buffer, 2 mM Lglutamine, and 25 μg/mL transferrin.)
adducts was inhibited by ascorbic acid or GSH but not by NAL (Figure 3a), indicating that radical intermediates of INH may be involved. Finally, INH was incubated with NAL in phosphate buffer that contains trace redox-active metals such as iron and copper, water (LC/MS grade), or in the presence of EDTA (10 μM) to elucidate the effect of the metals on INH’s auto-oxidation. The amount of IN-NAL adduct formed in water and in the presence of EDTA was significantly less than that in phosphate buffer alone, indicating that the trace metals present in phosphate buffer are able to catalyze the oxidation of INH to reactive species (Figure 3b). Formation of IN-NADP Conjugates in Human Liver Microsomes. An IN-NAL adduct was characterized previously in complete incubations of INH with human liver microsomes,10 and the formation of the adduct appeared to be P450-
and NADPH-dependent. In order to determine whether autooxidation of INH is a conspicuous pathway when compared to P450-mediated oxidation, incubations of INH with NAL in phosphate buffer or human liver microsomes with or without NADPH were performed. Notably, the presence of HLM alone increased the yield of the INH-NAL adduct. However, the additional presence of NADPH reduced the yield of the INNAL adduct (Figure 3c), probably due to preferential trapping of the reactive intermediates of INH by NADPH. The INNADP adduct was evidenced by the detection of a precursor ion at m/z 851.0 (Figure S1a, Supporting Information). A series of fragment ions, including m/z 429, m/z 410, m/z 312, and m/z 136, confirmed the presence of an NADP moiety in the adduct when compared to the MS/MS spectrum of 54
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Figure 4. Covalent modifications of HSA by INH in phosphate buffer, pH 7.4, and plasma in vitro. (a) INH-modified peptides were detected after human plasma and HSA (40 mg/mL) were incubated with INH (100 μM) for 24 h; (b) Multiple INH-modified peptides were detected after human plasma was incubated with 10 mM INH for 24 h.
residues and formed isonicotinic amide adducts. This finding demonstrated that auto-oxidation of INH can lead to covalent binding to proteins. We postulated that INH can be oxidized by oxygen dissolved in the phosphate buffer, with metal ion catalysis, to a reactive diazene, possibly via a hydrazyl radical.24 The diazene could react directly with nucleophiles to form covalent adducts. Alternatively, the diazene could decompose to an acyl radical in the presence of oxygen,9,22,25 leading to covalent binding (Scheme 1). Both pathways would result in the same adducts, the isonicotinic amide adducts with lysine residues in HSA.
NADPH (Figure S1b, Supporting Information). Consistent with previous observations,7 an ion at m/z 228.1, corresponding to the product derived from hydrolysis of isonicotinoylated NADP, was also identified, providing further evidence for the formation of the IN-NADP adduct (Figure S1c, Supporting Information). Modification of HSA by INH in Plasma in Vitro and in Patients. In order to determine whether INH can form albumin adducts under physiological conditions, incubation of plasma with INH (median peak plasma concentration 26.5 μM) was performed at 37 °C. Isonicotinoyl-lysine adducts were not detectable after a 16 h incubation under those conditions. However, it was possible to detect the adducts after a 24 h incubation with 100 μM INH (Figure 4a). Furthermore, when plasma was spiked with a very high concentration of INH (10 mM), a plasma concentration exceeding even those reached in extreme cases of deliberate INH poisoning,20 the adducts were detectable after 1 h of incubation (Figure 4b). We next investigated whether the same lysine adducts are formed in patients receiving either a therapeutic or a prophylactic regimen. HSA was isolated from four patients receiving 300 mg of INH once daily, and INH-modified albumin was detected in all patients, with K190 and K525 being the most prominent modification sites (Table 1). MS/MS analysis revealed that the same isonicotinic amide adducts were formed between INH and lysine residues in HSA (Figure S3, Supporting Information). The same adducts were also detectable in plasma isolated from patients administered with INH without any ADRs.
Scheme 1. Proposed Pathways of the Covalent Binding of INH to Proteins
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The involvement of INH auto-oxidation in covalent binding was further characterized by incubating INH with HSA under oxygen-depleted conditions and in the presence of potential inhibitors (ascorbic acid, GSH, and NAL). The yield of the covalent adducts was dramatically reduced by deoxygenation, ascorbic acid or GSH, but not by NAL. One possible explanation for these findings is that deoxygenation or addition of radical inhibitors prevented the initial generation of radicals, thereby attenuating covalent binding. In contrast, NAL had little effect on covalent binding, probably due to its inability to inhibit radical formation and its weaker nucleophilicity in comparison with lysine residues in the binding pockets of proteins.26,27 These findings indicated that radical intermediates might be involved in the adduction of proteins by INH. In addition to oxygen dissolved in the buffer, our study supports previous findings, which found that trace amounts of transition metals28,29 in buffer play an important role in INH autooxidation to reactive intermediates.30 For instance, IN-NAL
DISCUSSION There has been considerable debate regarding the mechanisms of INH-induced liver injury. However, it is generally accepted that bioactivation of INH mediated by P450 might play an important role in the hepatotoxicity.2,3,21,22 The reactive intermediates can either induce oxidative stress23 or bind to proteins,10 and either reaction may lead to hepatotoxicity. Several reactive intermediates of INH, diazene and diazohydroxide derivatives, the isonicotinoyl radical, and acetyl radicals, have been proposed as protein-haptenating species;8,12,13,22 however, it remains to be determined whether bioactivation is obligatory for the formation of INH-protein adducts. In this study, we found that INH formed albumin adducts in vitro in the absence of any oxidative enzymes or supplementary redox-active metals. When INH (10 mM) was incubated with HSA (40 mg/mL) in phosphate buffer at 37 °C for 16 h, LCMS/MS analysis revealed that INH was bound to lysine 55
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conjugate formation was almost completely inhibited by the addition of EDTA. The formation of the conjugate was also significantly suppressed when the experiment was conducted in high purity water. We next investigated the role of auto-oxidation and HLMmediated oxidation in the covalent binding of INH to protein. When INH was incubated with NAL with or without HLM/ NADPH, the abundance of the IN-NAL conjugate was about 3.5-times higher in HLM than that in phosphate buffer, suggesting that HLM-mediated oxidation may contribute dominantly to covalent binding. However, the relative contributions of auto-oxidation and HLM-mediated oxidation to INH covalent binding in vivo may differ from those observed in in vitro experiments. INH’s auto-oxidation catalyzed by Mn2+ has been shown to yield the same reaction products as the catalase-peroxidase catalyzed reaction at a comparable rate.30 It is possible that the concentrations of Mn2+ in the body, and especially in plasma,31 which has not been fully elucidated, may influence INH covalent binding in vivo, particularly under aerobic conditions. In contrast to HLM alone, NADPH significantly depressed the formation of IN-NAL conjugates. INH is known to form an isonicotinic acyl-NAD adduct that targets the Mycobacterium smegmatis InhA reductase, thereby inhibiting essential mycolic acid biosynthesis, effectively preventing cell capsule synthesis.32−34 It has been proposed that the formation of IN-NAD adducts involves the addition of either an isonicotinic acyl anion to NAD+ or an isonicotinic acyl radical to an NAD radical (Scheme 2).33 It is therefore plausible that NADPH in microsomal incubations acts as the dominant acyl radical scavenger and thereby blocks the formation of the NAL adduct. Notably, all the experiments to investigate the auto-oxidation of INH were carried out with high concentrations of INH (100 μM to 10 mM) that are much higher than therapeutic plasma concentrations (median Cmax in poor acetylators, 26.5 μM).18 Importantly, for excluding the detection of ex vivo protein modifications, no HSA adducts were detected when plasma was incubated with INH (26.5 μM) under physiological conditions for 24 h. However, three INH-modified lysine residues of albumin were detected in all patients administered INH in combination with other tuberculosis medications (Table 1). Considering the relatively long half-life of HSA in vivo (21 days)35 and the multiple doses of isoniazid taken by patients, it is plausible that INH-modified HSA would have accumulated in the plasma, leading to a higher level of modification than that occurring in vitro. The following steps were taken to ensure that the detected covalent modification of albumin in patients actually occurred in vivo and was not an ex vivo artifact. First, the blood samples were taken at least 24 h postdosing to ensure that INH had been cleared as the half-life of INH is in the range of 1.2−3.3 h;18 second, plasma was isolated rapidly (within 30 min following the withdrawal of blood) and stored at −80 °C (INH is stable in human plasma at ≤ −70 °C);36 third, in vitro incubation of plasma with the reported peak therapeutic concentration of INH (26.5 μM)18 showed that no adducts could be detected within 5 h (Figure S3, Supporting Information). MS/MS analysis revealed that the same isonicotinic amide adducts were formed between INH and lysine residues in HSA. These adducts could be formed in the liver, via relatively stable INH reactive metabolites generated by P450, and are then circulated in the blood. Alternatively, they could be formed in the blood from INH via auto-oxidation.
Scheme 2. Proposed Pathways for the Formation of INHNADP Adducts from Reactive Intermediates of INH Generated in Microsomal Incubationsa
a
Adapted from the pathways proposed by Rozwarski et al. for the formation in M. tuberculosis of an isonicotinic acyl-NADH adduct that inhibits InhA. Adapted with permission from ref 33. Copyright 1998 AAAS.
In conclusion, we have shown that INH binds to HSA via an auto-oxidation pathway in vitro, forming isonicotinic amide adducts with lysine residues in HSA. The same adducts were identified in patients’ plasma, indicating the auto-oxidation pathway may also contribute to the covalent binding of INH in vivo. We are now investigating how INH covalent adducts activate the humeral and cellular immune response in patients with DILI.
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ASSOCIATED CONTENT
* Supporting Information S
Formation of isoniazid NADP and protein adducts. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK. Tel: (+) 44 151 7945559. Fax: (+) 44 151 7945540. E-mail: [email protected]. Funding
This work was supported by the Medical Research Council Centre for Drug Safety Science (Grant Number MR/L006758/ 1). Notes
The authors declare no competing financial interest. 56
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(14) Koen, Y., Galeva, N., Hanzlik, R., Metushi, I., Uetrecht, J., Lu, J., and Watkins, P. (2014) Identification of protein targets for isoniazid reactive metabolites, https://hbc.ku.edu/abstracts-d1-d4-2014 (Oct 1, 2014). (15) Jones, R. P., Sutton, P., Greensmith, R. M., Santoyo-Castelazo, A., Carr, D. F., Jenkins, R., Rowe, C., Hamlett, J., Park, B. K., Terlizzo, M., O’Grady, E., Ghaneh, P., Fenwick, S. W., Malik, H. Z., Poston, G. J., and Kitteringham, N. R. (2013) Hepatic activation of irinotecan predicts tumour response in patients with colorectal liver metastases treated with DEBIRI: exploratory findings from a phase II study. Cancer Chemother. Pharmacol. 72, 359−368. (16) Meng, X., Howarth, A., Earnshaw, C. J., Jenkins, R. E., French, N. S., Back, D. J., Naisbitt, D. J., and Park, B. K. (2013) Detection of drug bioactivation in vivo: mechanism of nevirapine-albumin conjugate formation in patients. Chem. Res. Toxicol. 26, 575−583. (17) Greenough, C., Jenkins, R. E., Kitteringham, N. R., Pirmohamed, M., Park, B. K., and Pennington, S. R. (2004) A method for the rapid depletion of albumin and immunoglobulin from human plasma. Proteomics 4, 3107−3111. (18) Peloquin, C. A., Jaresko, G. S., Yong, C. L., Keung, A. C., Bulpitt, A. E., and Jelliffe, R. W. (1997) Population pharmacokinetic modeling of isoniazid, rifampin, and pyrazinamide. Antimicrob. Agents Chemother. 41, 2670−2679. (19) Meng, X., Jenkins, R. E., Berry, N. G., Maggs, J. L., Farrell, J., Lane, C. S., Stachulski, A. V., French, N. S., Naisbitt, D. J., Pirmohamed, M., and Park, B. K. (2011) Direct evidence for the formation of diastereoisomeric benzylpenicilloyl haptens from benzylpenicillin and benzylpenicillenic acid in patients. J. Pharmacol. Exp. Ther. 338, 841−849. (20) Alvarez, F. G., and Guntupalli, K. K. (1995) Isoniazid overdose: four case reports and review of the literature. Intensive Care Med. 21, 641−644. (21) Timbrell, J. A., Mitchell, J. R., Snodgrass, W. R., and Nelson, S. D. (1980) Isoniazid hepatoxicity: the relationship between covalent binding and metabolism in vivo. J. Pharmacol. Exp. Ther. 213, 364− 369. (22) Nelson, S. D., Mitchell, J. R., Timbrell, J. A., Snodgrass, W. R., and Corcoran, G. B., III (1976) Isoniazid and iproniazid: activation of metabolites to toxic intermediates in man and rat. Science 193, 901− 903. (23) Shuhendler, A. J., Pu, K., Cui, L., Uetrecht, J. P., and Rao, J. (2014) Real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing. Nat. Biotechnol. 32, 373−380. (24) Wengenack, N. L., and Rusnak, F. (2001) Evidence for isoniazid-dependent free radical generation catalyzed by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T). Biochemistry 40, 8990−8996. (25) Vanderwalt, B. J., Vanzyl, J. M., and Kriegler, A. (1994) Different oxidative pathways of isonicotinic-acid hydrazide and its meta-isomer, nicotinic-acid hydrazide. Int. J. Biochem. 26, 1081−1093. (26) Jenkins, R. E., Meng, X., Elliott, V. L., Kitteringham, N. R., Pirmohamed, M., and Park, B. K. (2009) Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin haptenated HSA in vitro and in vivo. Proteomics: Clin. Appl. 3, 720−729. (27) Hartman, F. C., Milanez, S., and Lee, E. H. (1985) Ionization constants of two active-site lysyl ε-amino groups of ribulosebisphosphate carboxylase/oxygenase. J. Biol. Chem. 260, 13968−13975. (28) Winder, F. G., and Denneny, J. M. (1959) Metal-catalysed autooxidation of isoniazid. Biochem. J. 73, 500−507. (29) Buettner, G. R. (1986) Ascorbate autoxidation in the presence of iron and copper chelates. Free Radical Res. Commun. 1, 349−353. (30) Bodiguel, J., Nagy, J. M., Brown, K. A., and Jamart-Gregoire, B. (2001) Oxidation of isoniazid by manganese and Mycobacterium tuberculosis catalase-peroxidase yields a new mechanism of activation. J. Am. Chem. Soc. 123, 3832−3833. (31) Rukgauer, M., Klein, J., and Kruse-Jarres, J. D. (1997) Reference values for the trace elements copper, manganese, selenium, and zinc in the serum/plasma of children, adolescents, and adults. J. Trace Elem. Med. Biol. 11, 92−98.
ACKNOWLEDGMENTS We thank Amy Driffill, Research Nurse, who helped collect the samples at St. James’s Hospital, Leeds, and the patients who participated in this project
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ABBREVIATIONS INH, isoniazid; IN, isonicotinic; DILI, drug-induced liver injury; ALT, alanine aminotransferase; INA, isonicotinic acid; NAL, N-α-acetyl-lysine; HLM, human liver microsomes; LCMS/MS, liquid chromatography−tandem mass spectrometry; MIF, macrophage migration inhibitory factor; HSA, human serum albumin; LC-MS, liquid chromatography−mass spectrometry; ADR, adverse drug reactions; RIF, rifampicin; PZA, pyrazinamide; ETB, ethambutol; MRM, multiple reaction monitoring
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REFERENCES
(1) Ng, C. S., Hasnat, A., Al Maruf, A., Ahmed, M. U., Pirmohamed, M., Day, C. P., Aithal, G. P., and Daly, A. K. (2014) N-acetyltransferase 2 (NAT2) genotype as a risk factor for development of drug-induced liver injury relating to antituberculosis drug treatment in a mixedethnicity patient group. Eur. J. Clin. Pharmacol. 70, 1079−1086. (2) Metushi, I. G., Cai, P., Zhu, X., Nakagawa, T., and Uetrecht, J. P. (2011) A fresh look at the mechanism of isoniazid-induced hepatotoxicity. Clin. Pharmacol. Ther. 89, 911−914. (3) Warrington, R. J., Tse, K. S., Gorski, B. A., Schwenk, R., and Sehon, A. H. (1978) Evaluation of isoniazid-associated hepatitis by immunological tests. Clin. Exp. Immunol. 32, 97−104. (4) Black, M., Mitchell, J. R., Zimmerman, H. J., Ishak, K. G., and Epler, G. R. (1975) Isoniazid-associated hepatitis in 114 patients. Gastroenterology 69, 289−302. (5) Metushi, I. G., Sanders, C., The Acute Liver Study, G., Lee, W. M., and Uetrecht, J. (2014) Detection of anti-isoniazid and anticytochrome P450 antibodies in patients with isoniazid-induced liver failure. Hepatology 59, 1084−1093. (6) Li, F., Miao, Y., Zhang, L., Neuenswander, S. A., Douglas, J. T., and Ma, X. (2011) Metabolomic analysis reveals novel isoniazid metabolites and hydrazones in human urine. Drug. Metab. Pharmacokinet. 26, 569−576. (7) Mahapatra, S., Woolhiser, L. K., Lenaerts, A. J., Johnson, J. L., Eisenach, K. D., Joloba, M. L., Boom, W. H., and Belisle, J. T. (2012) A novel metabolite of antituberculosis therapy demonstrates host activation of isoniazid and formation of the isoniazid-NAD+ adduct. Antimicrob. Agents Chemother. 56, 28−35. (8) Timbrell, J. A. (1979) The role of metabolism in the hepatotoxicity of isoniazid and iproniazid. Drug. Metab. Rev. 10, 125−147. (9) Amos, R. I., Gourlay, B. S., Yates, B. F., Schiesser, C. H., Lewis, T. W., and Smith, J. A. (2013) Mechanistic investigation of the oxidation of hydrazides: implications for the activation of the TB drug isoniazid. Org. Biomol. Chem. 11, 170−176. (10) Metushi, I. G., Nakagawa, T., and Uetrecht, J. (2012) Direct oxidation and covalent binding of isoniazid to rodent liver and human hepatic microsomes: humans are more like mice than rats. Chem. Res. Toxicol. 25, 2567−2576. (11) Li, F., Lu, J., Cheng, J., Wang, L., Matsubara, T., Csanaky, I. L., Klaassen, C. D., Gonzalez, F. J., and Ma, X. (2013) Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid cotherapy. Nat. Med. 19, 418−420. (12) Liu, K., Li, F., Lu, J., Gao, Z., Klaassen, C. D., and Ma, X. (2014) Role of CYP3A in isoniazid metabolism in vivo. Drug. Metab. Pharmacokinet. 29, 219−222. (13) Lu, J., Metushi, I., Uetrecht, J., Einhorn, S., Mann, D. A., Hanzlik, R. P., Watkins, P. B., and LeCluyse, E. L. (2014) Investigation of isoniazid DILI mechanisms in human induced pluripotent stem cell derived hepatocytes, www.cellulardynamics.com/issx/ CD|131001|SSX01.pdf (Oct 1, 2014). 57
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(32) Rawat, R., Whitty, A., and Tonge, P. J. (2003) The isoniazidNAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance. Proc. Natl. Acad. Sci. U.S.A. 100, 13881−13886. (33) Rozwarski, D. A., Grant, G. A., Barton, D. H. R., Jacobs, W. R., and Sacchettini, J. C. (1998) Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279, 98−102. (34) Vilcheze, C., Wang, F., Arai, M., Hazbon, M. H., Colangeli, R., Kremer, L., Weisbrod, T. R., Alland, D., Sacchettini, J. C., and Jacobs, W. R. (2006) Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid. Nat. Med. 12, 1027− 1029. (35) Muller, D., Karle, A., Meissburger, B., Hofig, I., Stork, R., and Kontermann, R. E. (2007) Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. J. Biol. Chem. 282, 12650−12660. (36) Hutchings, A., Monie, R. D., Spragg, B., and Routledge, P. A. (1983) A method to prevent the loss of isoniazid and acetylisoniazid in human plasma. Br. J. Clin. Pharmacol. 15, 263−266.
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Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin Xiaoli Meng,† James L. Maggs,† Toru Usui,† Paul Whitaker,‡ Neil S. French,† Dean J. Naisbitt,† and B. Kevin Park*,† †
MRC Center for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool L69 3GE, United Kingdom ‡ The Department of Respiratory Medicine, St. James’s Hospital, Leeds LS9 7TF, West Yorkshire, United Kingdom S Supporting Information *
ABSTRACT: Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.
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conjugate10 in complete microsomal incubations. These observations suggest that protein adduction might occur principally by acylation of the side chains of lysine residues. Direct confirmation that INH-protein adducts are formed in cells was achieved recently by Lu et al. using proteomic liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis.13 They identified isonicotinoylated prohibitin 2 and macrophage migration inhibitory factor, acylated at a lysine and proline residue, respectively, in human induced pluripotent stem cell derived hepatocytes. Similar analysis of S9 fractions of livers from INH-treated rats and mice identified D-dopachrome decarboxylase with an IN-lysine residue.14 However, none of these studies have investigated the covalent binding of INH to proteins in the absence of P450, nor have these adducts been detected in human patients exposed to INH therapy. Therefore, the aims of this study were to investigate the nature of INHprotein adducts formed in vitro by using human serum albumin (HSA) as a model protein and to characterize the INH-protein adducts formed in patients taking INH therapy.
INTRODUCTION Isoniazid (INH) remains a first line drug for the treatment of tuberculosis, even though its use is associated with the development of drug-induced liver injury (DILI).1 It has been reported that liver dysfunction occurs in 18−20% of patients receiving INH, but the majority of cases are mild reactions. It has been estimated that overt hepatotoxicity occurs in up to 1% of patients.2−4 The mechanisms underlying INH DILI remain ill-defined; however, there is evidence that the immune system may be involved. For instance, T cells from patients with INH DILI have been shown to proliferate ex vivo in the presence of INH and INH-modified proteins.3 In addition, antibodies against INH-modified proteins have been detected in patients with INH DILI.5 Together, these studies indicate that INHmodified proteins play an important role in the activation of a drug-specific immune response. INH is transformed to form structurally diverse stable metabolites in humans, including acetylhydrazine, isonicotinic acid (INA), and isonicotinic amide, which are potential products or precursors of chemically reactive species.2,6−8 It has been long debated on which reactive intermediates and pathways are involved in the formation of INH-protein adducts and which structural types of adduct are formed. Previous 14Clabeling studies have shown that acetylhydrazine contributed to covalent binding through acetyl radicals.8 Conversely, recent studies by Metushi et al. have proposed that INH could bind to hepatic microsomal proteins through a P450-generated acyl diazene (acyl diimide)9 or diazohydroxide intermediate.10 A reactive metabolite of INH is trapped as an isonicotinic amide of N-α-acetyl-lysine (NAL)10−12 but not as a glutathione © 2014 American Chemical Society
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EXPERIMENTAL PROCEDURES
Chemicals. HSA (97−99% pure) and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO), HLM was prepared from an adult liver sample obtained from a local hospital (Aintree University Hospital NHS Foundation Trust) with informed consent under ethical approval.15 Trypsin was from Promega (Madison, WI), liquid chromatography−mass spectrometry (LC-MS) grade solvents Received: July 12, 2014 Published: November 27, 2014 51
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Table 1. INH-Modified HSA Tryptic Peptides Detected in Patientsa modified lysine residues patient
gender
age
diagnosis
055
F
36
tuberculous lymphadenitis
056
F
60
070
F
63
081
M
81
chemoprophylaxis for antiTNF drugs chemoprophylaxis for antiTNF drugs pulmonary tuberculosis
a
INH dose 300 mg daily 300 mg daily 300 mg daily 300 mg daily
medication
adverse reactions
K137
K190
K525
INH/RIF/ PZA/ETB INH/RIF
cutaneous reactions after 48 h
−
+
+
elevated ALT of 502 U/L, 6 weeks into treatment
−
+
+
INH/RIF
elevated ALT of 347 U/L, 4 weeks into treatment
+
+
+
INH/RIF/ PZA/ETB
elevated ALT of 588 U/L and cutaneous reactions after 4 weeks of treatment
+
+
+
The drug-modified (isonicotinoylated) tryptic peptides of HSA isolated from patients’ plasma were characterized by LC-MS/MS. phosphate buffer, HLM (suspended in phosphate buffer; final protein concentration, 1 mg/mL), or HLM in the presence of NADPH (0.5 mM) were incubated with NAL (1 mM) at 37 °C for 3 h. The control experiment was performed by incubation of INH with HLM and NADPH in the absence of NAL under the same conditions. At the end of the incubation, bovine serum albumin (final concentration, 1 mg/ mL) was added to the mixture in phosphate buffer to remove components and products of the incubation that might cause matrix effects during LC-MS analyses. All samples were spiked with pyronaridine (final concentration, 100 nM) as an internal standard and extracted immediately with cold methanol. The extracts were evaporated to dry residues, reconstituted in 2% acetonitrile/0.1% formic acid and analyzed by LC-MS. Mass Spectrometric Characterization of INH NAL or NADP Conjugates. Samples were delivered into a 4000 QTRAP (AB Sciex, Framingham, MA,) coupled with a 1260 Infinity Quaternary Pump HPLC system (Agilent Technologies, Santa Clara, CA) and Kinetex C18 column (2.6 μm C18, 100 mm × 2.1 mm, Phenomenex, Macclesfield, Cheshire, U.K.). A gradient from 1% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 12 min was applied at a flow rate of 150 μL/min. Mass spectral analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) triggered enhanced product ion scan for quantitative analysis. The ionspray voltage was set to 4.5 kV, and the source temperature was maintained at 450 °C. High purity nitrogen was used for the GAS 1, GAS 2, curtain, and collision gases. The MRM transitions for INH conjugates were as follows: m/z 294 → 106 and m/z 294 → 189 for NAL conjugates and m/z 850.8 → 228 and 850.8 → 136 for NADP conjugates. Relative quantification of INH conjugates was performed by comparing the intensities of MRM peaks normalized against an internal standard (pyronaridine; m/z 518 → 447). Data were analyzed using Analyst software, version 1.5.1 (AB Sciex). Isolation of HSA from Plasma by Affinity Chromatography. HSA was isolated from patients’ plasma by affinity chromatography as described previously17 with slight modifications. An Affinity Removal Column HSA Only (Agilent Technologies) attached to a Vision Workstation (Applied Biosystems) was used to affinity-capture HSA from 50 μL of freshly thawed plasma. The HSA was eluted with 12 mM HCl and precipitated with 9 volumes of ice-cold methanol. The eluted protein was processed for LC-MS/MS analysis as described above. Covalent Binding of INH to HSA in Human Plasma in Vitro. Plasma from healthy volunteers was incubated with INH (freshly dissolved in potassium phosphate buffer) at concentrations of 26.5 μM (3.64 μg/mL, median Cmax plasma concentration in slow acetylators),18 100 μM, or 10 mM at 37 °C. Aliquots of 50 μL were removed after 0.5, 1, 2, 3, 15, and 24 h and processed for LC-MS/MS analysis as described above. Mass Spectrometric Characterization of INH-Modified HSA. Samples were reconstituted in 2% acetonitrile/0.1% formic acid (v/v), and aliquots of 2.4−5 pmol of tryptic digests were delivered into a QTRAP 5500 (AB Sciex) fitted with a NanoSpray II source by automated in-line liquid chromatography (U3000 HPLC System, 5 mm C18 nanoprecolumn and 75 μm × 15 cm C18 PepMap column;
were from Fisher Scientific UK Ltd. (Loughborough, Leicestershire), and all other standard reagents were from Sigma-Aldrich. Patients. Plasma samples were isolated from four patients with hepatic and/or cutaneous adverse drug reactions (ADRs) to INH, and stored immediately at −80 °C. Table 1 summarizes the patients’ demographics and the clinical features of the ADRs. Plasma samples from four patients administered with INH without ADRs were also included in this study. Approval for the study was acquired from the Liverpool and Leeds local research ethics committees; informed written consent was obtained from each patient. Blank plasma was obtained from healthy volunteers who gave informed consent according to a procedure approved by the University of Liverpool Committee of Research Ethics. Concentration-Dependent Modification of HSA by INH in Vitro. INH freshly dissolved in potassium phosphate buffer (10 mM, pH 7.4) was incubated with HSA (0.6 mM, 50 μL) or a serumsupplemented cell culture medium (R9 medium: RPMI 1640 supplemented with 10% pooled heat-inactivated human AB serum, 25 mM HEPES buffer, 2 mM L-glutamine, and 25 μg/mL transferrin) in sealed Eppendorf tubes at 37 °C for 16 h. The molar ratios of INH to protein were 0.1:1, 1:1, 10:1, and 20:1. Protein was precipitated twice with 9 volumes of ice-cold methanol to remove noncovalently bound INH, resuspended in 50 μL of phosphate buffer, and then reduced with 10 mM dithiothreitol (15 min) and alkylated with 55 mM iodoacetamide (15 min) at room temperature. The protein was precipitated with methanol once more and finally dissolved in 100 μL of 50 mM ammonium hydrogen carbonate, and 165 μg (1.25 nmol) of protein was digested with 1.6 μg of trypsin overnight at 37 °C. The samples were processed for LC-MS/MS analysis as described previously.16 Time-Dependent Modification of HSA by INH in Vitro. INH freshly dissolved in potassium phosphate buffer (10 mM, pH 7.4) was incubated with HSA (0.6 mM, 300 μL) at 37 °C (final INH concentration, 6 mM). Aliquots of 50 μL were removed after 0.5, 1, 2, 3, 16, and 24 h and processed for LC-MS/MS analysis as described above. Inhibition of Covalent Binding of INH to HSA. INH (6 mM) was incubated with HSA (0.6 mM, 50 μL) at 37 °C in either phosphate buffer (10 mM, pH 7.4) or phosphate buffer that had been purged with nitrogen for at least 10 min immediately beforehand. Another set of incubations was prepared from buffered solutions of INH (6 mM) and HSA (0.6 mM, 50 μL) spiked with ascorbic acid, GSH, or NAL at 0.01 mM, 0.1 mM, and 1 mM. At the end of the incubation (16 h), protein was precipitated twice with 9 volumes of ice-cold methanol to remove noncovalently bound drugs and processed for LC-MS/MS analysis as described above. Formation of IN-NAL Conjugates in Various Media. Freshly prepared INH solution (10 mM, in either LC-MS grade water, or phosphate buffer, phosphate buffer containing EDTA (10 μM), or phosphate buffer purged with nitrogen for at least 10 min was incubated with NAL (10 mM) at 37 °C for 16 h. The resulting products were spiked with 50 nM pyronaridine as an internal standard and analyzed by LC-MS. Formation of IN-NADP Conjugates in Human Liver Microsomes. Freshly prepared solutions of INH (100 μM or 1 mM) in 52
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Figure 1. LC-MS/MS analysis of an INH-modified albumin peptide identified in incubations with HSA, R9 medium, and plasma. (a) MS/MS spectrum of the INH-modified tryptic peptide 182LDELRDEGK(INA)ASSAK195 with a mass addition of 105 amu; (b) proposed pathway for the generation of the fragment ion of INA-lysine at m/z 189 by loss of CO and NH3. Dionex, Sunnyvale, CA) via a 10 μm inner diameter PicoTip (New Objective, Woburn, MA). A gradient from 2% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 60 min was applied at a flow rate of 300 nL/min. The ion spray potential was set to 2,200−3,500 V, the nebulizer gas to 19, and the interface heater to 150 °C. MRM transitions specific for INH-modified peptides were selected as follows: the m/z values were calculated for all possible peptides containing a lysine residue together with the characteristic fragment ion of m/z 189. MRM transitions were acquired at unit resolution in both the Q1 and Q3 quadrupoles to maximize specificity. They were optimized for collision energy and collision cell exit potential, and the dwell time was 20 ms. MRM survey scans were used to trigger enhanced product ion scans of INH-modified peptides, with Q1 set to unit resolution, dynamic fill selected, and dynamic exclusion for 20 s. Total ion counts were determined from a second aliquot of each sample analyzed by conventional LC-MS and were used to normalize sample loading on the column. Relative quantification of modified peptides was performed by comparing the relative normalized intensity of MRM peaks for each of the modified residues against total ion counts across samples. Data were analyzed using Analyst software (AB Sciex). Statistical Analysis. Data are presented as the means and their standard deviations. Statistical comparisons of experimental groups were made using the paired t tests method (Sigmaplot 12 software, Systat software Inc., San Jose, CA). A value of P < 0.05 was considered to be significant.
of any oxidative enzymes or supplementary redox-active metals. INH modification was evidenced by the MS/MS spectrum shown in Figure 1a.The isonicotinic amide adduct of Lys190 resulted in a doubly charged ion at m/z 812.5, corresponding to a mass increase of 105 amu to the peptide 182LDELRDEGKASSAK195. The peptide sequence was confirmed by a series of b and y product ions, and the site of modification was confirmed by the presence of a9* + 2 (m/z 567.1), b9* + 2 (m/z 581.1), and y6* + 2 (m/z 694.9). A characteristic fragment ion at m/z 189 amu was also observed, corresponding to the INH adducted lysine immonium ion with the loss of an ammonia molecule (Figure 1b). The INH modification appeared to be concentration- and time-dependent (Figure 2a and b). A semiquantitative analysis of modification at each site was performed by determining the peak area for the extracted masses of the modified peptides, followed by normalization of the ion intensity against the internal standard. A concentration- and time-dependent increase in normalized ion counts was observed for the modified K137, K212, and K525 peptides (Figure 2a and b). Furthermore, when INH was incubated with a serumsupplemented cell culture medium, a similar profile of lysine adduction was observed (Figure 2c). Inhibition of the Auto-oxidation of INH in Vitro. In order to further elucidate the auto-oxidation of INH to reactive intermediates, the following experiments were performed. First, INH was incubated with HSA at a molar ratio of 10:1 in phosphate buffer or buffer that had been purged with nitrogen. The yield of the covalent protein adducts was dramatically reduced by the expulsion of dissolved oxygen (Figure 3a). Second, ascorbic acid, GSH, and NAL were coincubated individually with INH and HSA. The formation of the covalent
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RESULTS Characterization of INH-Modified HSA in Vitro. To probe the chemical basis of INH’s covalent binding to HSA, INH was incubated directly with HSA in phosphate buffer (10 mM and pH 7.4). After 16 h, the protein was processed and digested with trypsin as described previously.19 The resulting tryptic peptides were analyzed by LC-MS/MS. Surprisingly, INH-modified peptides were readily detectable in the absence 53
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Figure 3. Characteristics of the spontaneous chemical activation of INH in vitro. (a) Covalent binding of INH to HSA was inhibited by purging oxygen from the solution with nitrogen and by ascorbic acid (ASC) and GSH. (b) Conjugation of INH with NAL was oxygendependent and catalyzed by the trace, redox-active, EDTA chelatable metals present in phosphate buffer (PB). (c) The IN-NAL conjugate was formed in phosphate buffer, in the presence of human liver microsomes (1 mg/mL) without NADPH, or with NADPH (0.5 mM); the control experiment was performed by incubation of INH with HLM and NADPH in the absence of NAL. Data are expressed as the means ± SDs from three different experiments. Statistical comparisons of groups were determined by use of paired t tests; *p < 0.05 was considered to be significant.
Figure 2. LC-MS/MS analysis of INH-modified albumin peptides identified via in vitro incubation with HSA and R9 medium. Modification of some of the lysine residues by INH in phosphate buffer was (a) concentration dependent and (b) time dependent (INH/HSA molar ration at 10:1). (c) Similar lysine adduct profiles were observed when HSA and R9 medium were incubated with INH. (R9 medium: RPMI 1640 supplemented with 10% pooled heat inactivated human AB serum, 25 mM HEPES buffer, 2 mM Lglutamine, and 25 μg/mL transferrin.)
adducts was inhibited by ascorbic acid or GSH but not by NAL (Figure 3a), indicating that radical intermediates of INH may be involved. Finally, INH was incubated with NAL in phosphate buffer that contains trace redox-active metals such as iron and copper, water (LC/MS grade), or in the presence of EDTA (10 μM) to elucidate the effect of the metals on INH’s auto-oxidation. The amount of IN-NAL adduct formed in water and in the presence of EDTA was significantly less than that in phosphate buffer alone, indicating that the trace metals present in phosphate buffer are able to catalyze the oxidation of INH to reactive species (Figure 3b). Formation of IN-NADP Conjugates in Human Liver Microsomes. An IN-NAL adduct was characterized previously in complete incubations of INH with human liver microsomes,10 and the formation of the adduct appeared to be P450-
and NADPH-dependent. In order to determine whether autooxidation of INH is a conspicuous pathway when compared to P450-mediated oxidation, incubations of INH with NAL in phosphate buffer or human liver microsomes with or without NADPH were performed. Notably, the presence of HLM alone increased the yield of the INH-NAL adduct. However, the additional presence of NADPH reduced the yield of the INNAL adduct (Figure 3c), probably due to preferential trapping of the reactive intermediates of INH by NADPH. The INNADP adduct was evidenced by the detection of a precursor ion at m/z 851.0 (Figure S1a, Supporting Information). A series of fragment ions, including m/z 429, m/z 410, m/z 312, and m/z 136, confirmed the presence of an NADP moiety in the adduct when compared to the MS/MS spectrum of 54
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Figure 4. Covalent modifications of HSA by INH in phosphate buffer, pH 7.4, and plasma in vitro. (a) INH-modified peptides were detected after human plasma and HSA (40 mg/mL) were incubated with INH (100 μM) for 24 h; (b) Multiple INH-modified peptides were detected after human plasma was incubated with 10 mM INH for 24 h.
residues and formed isonicotinic amide adducts. This finding demonstrated that auto-oxidation of INH can lead to covalent binding to proteins. We postulated that INH can be oxidized by oxygen dissolved in the phosphate buffer, with metal ion catalysis, to a reactive diazene, possibly via a hydrazyl radical.24 The diazene could react directly with nucleophiles to form covalent adducts. Alternatively, the diazene could decompose to an acyl radical in the presence of oxygen,9,22,25 leading to covalent binding (Scheme 1). Both pathways would result in the same adducts, the isonicotinic amide adducts with lysine residues in HSA.
NADPH (Figure S1b, Supporting Information). Consistent with previous observations,7 an ion at m/z 228.1, corresponding to the product derived from hydrolysis of isonicotinoylated NADP, was also identified, providing further evidence for the formation of the IN-NADP adduct (Figure S1c, Supporting Information). Modification of HSA by INH in Plasma in Vitro and in Patients. In order to determine whether INH can form albumin adducts under physiological conditions, incubation of plasma with INH (median peak plasma concentration 26.5 μM) was performed at 37 °C. Isonicotinoyl-lysine adducts were not detectable after a 16 h incubation under those conditions. However, it was possible to detect the adducts after a 24 h incubation with 100 μM INH (Figure 4a). Furthermore, when plasma was spiked with a very high concentration of INH (10 mM), a plasma concentration exceeding even those reached in extreme cases of deliberate INH poisoning,20 the adducts were detectable after 1 h of incubation (Figure 4b). We next investigated whether the same lysine adducts are formed in patients receiving either a therapeutic or a prophylactic regimen. HSA was isolated from four patients receiving 300 mg of INH once daily, and INH-modified albumin was detected in all patients, with K190 and K525 being the most prominent modification sites (Table 1). MS/MS analysis revealed that the same isonicotinic amide adducts were formed between INH and lysine residues in HSA (Figure S3, Supporting Information). The same adducts were also detectable in plasma isolated from patients administered with INH without any ADRs.
Scheme 1. Proposed Pathways of the Covalent Binding of INH to Proteins
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The involvement of INH auto-oxidation in covalent binding was further characterized by incubating INH with HSA under oxygen-depleted conditions and in the presence of potential inhibitors (ascorbic acid, GSH, and NAL). The yield of the covalent adducts was dramatically reduced by deoxygenation, ascorbic acid or GSH, but not by NAL. One possible explanation for these findings is that deoxygenation or addition of radical inhibitors prevented the initial generation of radicals, thereby attenuating covalent binding. In contrast, NAL had little effect on covalent binding, probably due to its inability to inhibit radical formation and its weaker nucleophilicity in comparison with lysine residues in the binding pockets of proteins.26,27 These findings indicated that radical intermediates might be involved in the adduction of proteins by INH. In addition to oxygen dissolved in the buffer, our study supports previous findings, which found that trace amounts of transition metals28,29 in buffer play an important role in INH autooxidation to reactive intermediates.30 For instance, IN-NAL
DISCUSSION There has been considerable debate regarding the mechanisms of INH-induced liver injury. However, it is generally accepted that bioactivation of INH mediated by P450 might play an important role in the hepatotoxicity.2,3,21,22 The reactive intermediates can either induce oxidative stress23 or bind to proteins,10 and either reaction may lead to hepatotoxicity. Several reactive intermediates of INH, diazene and diazohydroxide derivatives, the isonicotinoyl radical, and acetyl radicals, have been proposed as protein-haptenating species;8,12,13,22 however, it remains to be determined whether bioactivation is obligatory for the formation of INH-protein adducts. In this study, we found that INH formed albumin adducts in vitro in the absence of any oxidative enzymes or supplementary redox-active metals. When INH (10 mM) was incubated with HSA (40 mg/mL) in phosphate buffer at 37 °C for 16 h, LCMS/MS analysis revealed that INH was bound to lysine 55
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conjugate formation was almost completely inhibited by the addition of EDTA. The formation of the conjugate was also significantly suppressed when the experiment was conducted in high purity water. We next investigated the role of auto-oxidation and HLMmediated oxidation in the covalent binding of INH to protein. When INH was incubated with NAL with or without HLM/ NADPH, the abundance of the IN-NAL conjugate was about 3.5-times higher in HLM than that in phosphate buffer, suggesting that HLM-mediated oxidation may contribute dominantly to covalent binding. However, the relative contributions of auto-oxidation and HLM-mediated oxidation to INH covalent binding in vivo may differ from those observed in in vitro experiments. INH’s auto-oxidation catalyzed by Mn2+ has been shown to yield the same reaction products as the catalase-peroxidase catalyzed reaction at a comparable rate.30 It is possible that the concentrations of Mn2+ in the body, and especially in plasma,31 which has not been fully elucidated, may influence INH covalent binding in vivo, particularly under aerobic conditions. In contrast to HLM alone, NADPH significantly depressed the formation of IN-NAL conjugates. INH is known to form an isonicotinic acyl-NAD adduct that targets the Mycobacterium smegmatis InhA reductase, thereby inhibiting essential mycolic acid biosynthesis, effectively preventing cell capsule synthesis.32−34 It has been proposed that the formation of IN-NAD adducts involves the addition of either an isonicotinic acyl anion to NAD+ or an isonicotinic acyl radical to an NAD radical (Scheme 2).33 It is therefore plausible that NADPH in microsomal incubations acts as the dominant acyl radical scavenger and thereby blocks the formation of the NAL adduct. Notably, all the experiments to investigate the auto-oxidation of INH were carried out with high concentrations of INH (100 μM to 10 mM) that are much higher than therapeutic plasma concentrations (median Cmax in poor acetylators, 26.5 μM).18 Importantly, for excluding the detection of ex vivo protein modifications, no HSA adducts were detected when plasma was incubated with INH (26.5 μM) under physiological conditions for 24 h. However, three INH-modified lysine residues of albumin were detected in all patients administered INH in combination with other tuberculosis medications (Table 1). Considering the relatively long half-life of HSA in vivo (21 days)35 and the multiple doses of isoniazid taken by patients, it is plausible that INH-modified HSA would have accumulated in the plasma, leading to a higher level of modification than that occurring in vitro. The following steps were taken to ensure that the detected covalent modification of albumin in patients actually occurred in vivo and was not an ex vivo artifact. First, the blood samples were taken at least 24 h postdosing to ensure that INH had been cleared as the half-life of INH is in the range of 1.2−3.3 h;18 second, plasma was isolated rapidly (within 30 min following the withdrawal of blood) and stored at −80 °C (INH is stable in human plasma at ≤ −70 °C);36 third, in vitro incubation of plasma with the reported peak therapeutic concentration of INH (26.5 μM)18 showed that no adducts could be detected within 5 h (Figure S3, Supporting Information). MS/MS analysis revealed that the same isonicotinic amide adducts were formed between INH and lysine residues in HSA. These adducts could be formed in the liver, via relatively stable INH reactive metabolites generated by P450, and are then circulated in the blood. Alternatively, they could be formed in the blood from INH via auto-oxidation.
Scheme 2. Proposed Pathways for the Formation of INHNADP Adducts from Reactive Intermediates of INH Generated in Microsomal Incubationsa
a
Adapted from the pathways proposed by Rozwarski et al. for the formation in M. tuberculosis of an isonicotinic acyl-NADH adduct that inhibits InhA. Adapted with permission from ref 33. Copyright 1998 AAAS.
In conclusion, we have shown that INH binds to HSA via an auto-oxidation pathway in vitro, forming isonicotinic amide adducts with lysine residues in HSA. The same adducts were identified in patients’ plasma, indicating the auto-oxidation pathway may also contribute to the covalent binding of INH in vivo. We are now investigating how INH covalent adducts activate the humeral and cellular immune response in patients with DILI.
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ASSOCIATED CONTENT
* Supporting Information S
Formation of isoniazid NADP and protein adducts. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK. Tel: (+) 44 151 7945559. Fax: (+) 44 151 7945540. E-mail: [email protected]. Funding
This work was supported by the Medical Research Council Centre for Drug Safety Science (Grant Number MR/L006758/ 1). Notes
The authors declare no competing financial interest. 56
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ACKNOWLEDGMENTS We thank Amy Driffill, Research Nurse, who helped collect the samples at St. James’s Hospital, Leeds, and the patients who participated in this project
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ABBREVIATIONS INH, isoniazid; IN, isonicotinic; DILI, drug-induced liver injury; ALT, alanine aminotransferase; INA, isonicotinic acid; NAL, N-α-acetyl-lysine; HLM, human liver microsomes; LCMS/MS, liquid chromatography−tandem mass spectrometry; MIF, macrophage migration inhibitory factor; HSA, human serum albumin; LC-MS, liquid chromatography−mass spectrometry; ADR, adverse drug reactions; RIF, rifampicin; PZA, pyrazinamide; ETB, ethambutol; MRM, multiple reaction monitoring
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