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Division of Drug Discovery (Z.Y., N.M., R.T., G.C.L., G.W.C., N.H.), and Chemical & Pharmaceutical Development (H.M.Z.), Johnson & Johnson Pharmaceutical Research & Development, LLC, Spring House, Pennsylvania
(Received July 1, 2005; accepted September 16, 2005)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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), and acute exposure can lead to a number of toxic effects such as uremia (De Smet et al., 2003) and hepatotoxicity (Kamijo et al., 2003). Presumably, uremic toxicity of p-cresol results from its high serum protein-binding affinity. Because p-cresol competes with drugs for protein binding, it can substantially enhance the toxic effect of uremia (Lesaffer et al., 2001). In vitro studies have suggested a potential association between hepatotoxicity and metabolic activation of p-cresol (Thompson et al., 1994, 1995), although such a relationship remains to be established in vivo.
In rat liver microsomal incubations, it has been found that 4-hydroxybenzylalcohol is the primary metabolite of p-cresol (Sato et al., 1956). In vitro studies using rat liver microsomes have also demonstrated that p-cresol is oxidized to form a quinone methide intermediate that can be detected as a stable conjugate when glutathione (GSH) is present as a trapping agent in incubations (Thompson et al., 1995). The quinone methide is a highly reactive electrophile, which can alkylate cellular proteins and nucleic acids and thus potentially lead to toxic effects (Monks and Jones, 2002). However, a similar study has not been conducted using human liver microsomes.
In the present study we investigated the metabolism and bioactivation of p-cresol in human liver microsomes. Structural analysis of both stable and reactive metabolites revealed that, in addition to oxidation of the methyl group, oxidation of the benzene ring can lead to formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate as 4-methyl-ortho-benzoquinone. In addition, it was found that, in microsomal incubations, 4-hydroxybenzylalcohol derived from p-cresol is further oxidized to 4-hydroxybenzaldehyde.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-glutamyl-cystein-glycin-13C2-15N] was obtained from Cambridge Isotope Laboratories (Andover, MA), and isotopic purity was 94% as estimated by the supplier using NMR. Instrumentation. Mass spectrometry analyses were performed on a Micromass (Waters, Manchester, UK) Quattro Micro triple quadrupole mass spectrometer. NMR spectra were obtained on a Bruker (Newark, DE) DPX 300 NMR spectrometer, and chemical shifts of H1 were expressed relative to tetramethylsilane.
Microsomal Incubations and Stable Isotope Trapping of Reactive Metabolites. All incubations were performed at 37°C in a water bath as previously described (Yan et al., 2002). p-Cresol or its derivative was individually mixed with human or rat liver microsomal protein in 50 mM potassium phosphate buffer (pH 7.4). After a 5-min preincubation at 37°C, reactions were initiated by the addition of a NADPH-generating system to give a final volume of 1.0 ml. The final reaction mixture contained p-cresol at a desired concentration (10–200 µM), 1 mg/ml microsomal protein, 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride. After a 30-min incubation, reactions were terminated by the addition of 150 µl of trichloroacetic acid (10%). Samples were centrifuged at 10,000g for 15 min at 4°C to pellet the precipitated protein, and supernatants were subjected to LC-MS/MS for direct analysis of metabolites.
To trap reactive metabolites formed in microsomal incubations, 1 mM GSH or a mixture of GSH and GSX (1:1) was added to reaction mixtures (Yan and Caldwell, 2004). Reactions were initiated by the addition of the NADPH regeneration solution, and incubations were performed for 60 min.
To assess formation of GSH conjugates by individual P450s, incubations were performed similarly, except that liver microsomes were substituted by Supersomes that contained individual enzymes at a final concentration of 100 pmol/ml. To analyze the metabolite formed by individual P450s, GSH was omitted in the incubations. Resulting samples were processed as described above, and analyzed by LC-MS/MS.
Synthesis of 3-(Glutathione-S-yl)-5-methyl-ortho-hydroquinone. 4-Methyl-ortho-hydroquinone (0.62 g) in 10 ml of diethyl ether was converted to 4-methyl-[1,2]benzoquinone by slow addition to a diethyl ether solution of 3,4,5,6-tetrachloro-[1,2]benzoquinone (1.34 g) at –5–0°C (Carlson and Miller, 1985). The resulting reaction mixture was treated with glutathione (1.54 g) in situ at 0°C and followed by the addition of 20 ml of H2O and 10 ml of tetrahydrofuran. After stirring for 24 h at room temperature, the reaction mixture was diluted with 20 ml of H2O. The aqueous layer was washed with 30 ml of diethyl ether and lyophilized to give a crude solid product (2.01 g). The crude solid product was analyzed by LC-MS/MS for GSH adducts. A total amount of 50 mg of crude product was subjected to purification by a reversedphase HPLC to provide 28 mg of the GSH adduct. MS, m/z 429.9 (MH+); 1H NMR (CD3OD): 
2.17 (s, 3H); 2.14–2.23 (m, 2 H); 2.46–2.59 (m, 2H); 3.06 (dd, J = 13.8, 8.8 Hz, 1H); 3.30 (m, 1H); 3.86 (s, 2H); 4.02 (t, J = 6.6 Hz, 1H); 4.47 (m, 1H); 6.60 (br d, J = 1.6 Hz, 1H); 6.70 (br d, J = 1.6 Hz, 1H); 13C NMR (CD3OD): 174.5, 173.0, 172.7, 171.7, 146.4, 144.6, 130.8, 126.3, 120.4, 117.6, 54.6, 53.7, 41.8, 37.2, 32.5, 27.1, 20.7.
LC-MS/MS Analyses. LC-MS/MS analyses were performed on a Micromass (Waters) Quattro Micro triple quadrupole mass spectrometer that was interfaced to an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA). The electrospray ionization ion source was operated in either the positive ion mode for analyzing GSH adducts or the negative mode for detecting metabolites of p-cresol, and experimental parameters were set as follows: capillary voltage 3.2 kV, source temperature 120°C, desolvation temperature 300°C, sample cone voltage 22 V. Data were processed using the Masslynx version 4.0 software from Waters.
For rapid profiling of GSH adducts, samples were first subjected to chromatographic separations on an Agilent Zorbax SB C18 column (2.1 x 50 mm), and eluents were introduced to the triple quadrupole mass spectrometer, which was operated in the neutral loss (NL) scanning mode, detecting all protonated molecules losing 129 Da under collision-induced dissociation (CID). The starting mobile phase consisted of 95% water (0.5% acetic acid), and the metabolites were eluted using a single gradient of 95% water to 95% acetonitrile over 7 min at a flow rate of 0.25 ml/min. At 7 min, the column was flushed with 95% acetonitrile for 2 min before re-equilibration at initial conditions. LC-MS/MS analyses were carried out on 30-µl aliquots of samples. Mass spectra collected in the NL scanning mode were obtained by scanning over the range m/z 350 to 600 in 2 s.
For subsequent structural characterization of metabolites and GSH adducts, an Agilent Zorbax SB C18 column (2.1 x 150 mm) was used for chromatographic separations. A 20-min LC run was performed to separate metabolites: the starting mobile phase consisted of 95% water (0.5% acetic acid), and the metabolites were eluted using a single gradient of 95% water to 95% acetonitrile over 15 min at a flow rate of 0.25 ml/min. At 15 min, the column was flushed with 95% acetonitrile for 2 min before re-equilibration under initial conditions.
To relatively compare the level of GSH adducts and metabolites formed in incubations, the mass spectrometer was operated in the multiple reaction monitoring mode using collision energy at 20 eV. Multiple reaction monitoring transitions used are m/z 414
179 for glutathionyl-4-methylphenol, m/z 430301 for 4-(glutathione-S-yl)-5-methyl-ortho-hydroquinone, and m/z 12192 for 4-hydroxybenzaldehyde, respectively.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Reactive metabolites were trapped to form both natural and stable isotope-labeled GSH adducts. Samples were subjected to LC-MS/MS NL scan detecting protonated molecules that lost 129 Da upon CID. Because glutathione was triply labeled at glycine, formed GSH adducts were positively identified by examining a unique isotopic doublet of a mass difference of 3 Da (Yan and Caldwell, 2004). As shown in Fig. 1A, although several peaks showed responses to the NL scanning for 129 Da, four components, glutathionyl-4-methylphenol (GA) at 3.20 min, 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone (GB) at 3.96 min, GC at 4.2 min, and GD at 3.75 min, exhibited the expected isotopic doublet, respectively. Both GC and GD appeared as small shoulder peaks of GB under current chromatographic conditions (LC column, C18, 2.1 x 50 mm) but were well resolved on a longer column (C18, 2.1 x 150 mm) (data not shown). The protonated molecule of GA displayed an isotopic doublet at m/z 414 and 417 (Fig. 1B), suggesting formation of a GSH adduct likely resulting from the quinone methide intermediate (Thompson et al., 1995). Interestingly, GB, GC, and GD exhibited an identical isotopic doublet at m/z 430 and 433 (Fig. 1, C to E), suggesting formation of three additional isomeric GSH adducts. No GSH adducts were detected when either p-cresol or the NADPH regenerating system was omitted from incubations. These results suggested that GSH adducts were formed from reactive metabolites of p-cresol via P450-mediated metabolic pathways. In addition, all four adducts (GA–GD) were detected in both human and rat liver microsomes at different concentrations of p-cresol (10–200 µM), which indicated that formation of GSH adducts was not dependent upon substrate concentrations.
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-glutamate (129 Da), respectively, which further confirmed the presence of a GSH moiety in the metabolite; the product ion at m/z 107 was apparently derived from losing the GSH moiety of the conjugate.
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-glutamate were observed for all three metabolites, giving rise to product ions at m/z 355 and 301, respectively, which confirmed that GB, GC, and GD were isomeric GSH adducts.
To further elucidate the structure of GSH adducts, 4-methylphenol-2,3,5,6-d4 was used in microsomal incubations in the presence of GSH. NL MS/MS analysis revealed that precursor ions of GA appeared at m/z 418 (Fig. 3A). A mass shift of 4 Da observed for GA indicated that the glutathione moiety is attached to the methyl group, which is consistent with glutathionyl-4-methylphenol, previously proposed by others (Thompson et al., 1995). A mass shift of 2 Da was observed for GB, GC, and GD (Fig. 3, B to D), suggesting that both the GSH moiety and the hydroxyl group were on the aromatic ring. Therefore, it was concluded that those adducts (GB, GC, and GD) were not derived from hydroxylation of GA. Otherwise, a mass shift of 3 Da would be detected for those three conjugates.
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Identification of Stable and Reactive Metabolites of p-Cresol in Incubations. It can be envisioned that p-cresol was first converted to 4-methyl-ortho-hydroquinone, which was further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone, resulting in formation of isomeric adducts in the presence of GSH in incubations. To further confirm this bioactivation pathway, microsomal incubations of p-cresol were performed in the absence of GSH. LC-MS/MS analysis of microsomal samples detected two metabolites (M1 and M2) showing molecular ions ([M – H]–) at m/z 123 Da (Fig. 4A). M1 eluted at 7.8 min (Fig. 4B) was identified by tandem MS as 4-hydroxybenzylalcohol. As shown in Fig. 4C, molecular ion ([M – H]–) at m/z 123 lost a water to give product ions at m/z 105 that was subsequently fragmented to generate product ions at m/z 77 (C6H5–). The identity of M1 was further confirmed by comparing the LC-MS/MS characteristics of the reference compound.
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Another major metabolite M3 ([M – H]–, m/z 121, retention time 9.35 min) was detected in the same microsomal incubations (Fig. 5A). As seen in the CID-MS/MS spectrum of M3 (Fig. 5B), the most abundant product ions appeared at m/z 92, apparently resulting from a loss of CHO, which suggested that M3 is 4-hydroxybenzaldehyde. LC-MS/MS analysis of the reference compound further confirmed the structure of M3.
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The major conjugate was unambiguously identified as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone by comparing 1H NMR spectra in the aromatic region of the GSH conjugate and 4-methyl-ortho-hydroquinone. For 4-methyl-ortho-hydroquinone, couplings between two aromatic protons at meta-positions (Ha and Hb) were observed, with a coupling constant of 1.6 Hz (Fig. 7A). The NMR spectrum of the conjugate is shown in Fig. 7B, which clearly suggests that two aromatic protons are meta-positioned. In addition, high-resolution NMR revealed weak couplings between the aromatic protons and the aliphatic protons on the methyl group (data not shown). The results clearly indicated that the GSH moiety is at the C-3 position. If the GSH moiety were at the C-4 position, the meta-couplings would not be detected. Apparently, 6-(glutathione-S-yl)-5-methyl-benzene-1, 2-diol would exhibit two doublet peaks (J = 7.5 Hz), due to the coupling between two ortho aromatic protons (Hb and Hc). The addition of glutathione to 4-methyl-[1,2]benzoquinone could result in three adducts, 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone, 4-(glutathione-S-yl)-5-methyl-ortho-hydroquinone, and 6-(glutathione-S-yl)-5-methyl-ortho-hydroquinone. Two minor adducts were not characterized by NMR because of low abundance and difficulty in isolation. Presumably, they are 4-(glutathione-S-yl)-5-methyl-ortho-hydroquinone and 6-(glutathione-S-yl)-5-methyl-ortho-hydroquinone, respectively.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), the proof of the reactive metabolite as a causative agent for p-cresol-induced toxicity still remains elusive. It is essential to completely understand bioactivation pathways of p-cresol to comprehensively address the toxicity mechanism. In this study, we have investigated bioactivation of p-cresol in human liver microsomes fortified with NADPH, in an attempt to elucidate metabolic pathways leading to formation of reactive intermediates. In addition to the GSH adduct (GA) derived from the quinone methide intermediate, three new GSH adducts (GB, GC, and GD) were detected using stable isotope GSH trapping in combination with LC-MS/MS analysis of incubations of p-cresol with human liver microsomes. The major GSH adduct GB was identified as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone by tandem MS in combination with NMR analysis of the synthetic conjugate.
Based on our results, a new bioactivation mechanism of p-cresol has been proposed. As shown in Scheme 1, there are two metabolic pathways responsible for bioactivation of p-cresol. As previously proposed by others (Thompson et al., 1995), oxidation of the methyl group leads to formation of a quinone methide intermediate that is trapped by GSH to form glutathionyl-4-methylphenol (GA) in microsomal incubations. Formation of the quinone methide intermediate is mediated by several P450s which include CYP2D6, 2C19, 1A2, 1A1, and 2E1. The newly identified pathway is aromatic oxidation leading to formation of 4-methyl-ortho-hydroquinone that is further oxidized to 4-methyl-[1,2]benzoquinone. The o-quinone intermediate could be trapped in microsomal incubations by GSH to form three adducts, but 3-(glutathione-S-yl)-5-methyl ortho-hydroquinone is the predominant conjugate. This bioactivation pathway was further confirmed by incubating 4-methyl-ortho-hydroquinone with human microsomes to generate the same conjugates. In the presence of GSH as a trapping agent, three adducts were detected, which exhibited chromatographic and MS characteristics identical to those of conjugates formed by p-cresol. The aromatic oxidation pathway is primarily mediated by CYP2E1, and also to a lesser extent by other P450s such as CYP1A1, 1A2, and 2D6. Additionally, two new metabolites, 4-methyl-ortho-hydroquinone and 4-hydroxybenzaldehyde, were identified in microsomal incubations of p-cresol using LC-MS/MS. It is apparent that 4-hydroxybenzaldehyde is the oxidation metabolite of 4-hydroxybenzylalcohol, the primary metabolite of p-cresol (Scheme 1). Formation of 4-hydroxybenzaldehyde is primarily catalyzed by 1A2, and other isozymes such as 1A1 and 2D6 are also active.
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). It is reasonable to expect that p-cresol can also be oxidized by P450s to 4-methyl-ortho-hydroquinone, which is subsequently converted to a reactive o-quinone intermediate. This bioactivation pathway has not been reported in previous studies using rat liver microsomes (Thompson et al., 1994, 1995). The reason why this metabolic pathway was not identified previously by other investigators is not clear. Species difference is not a factor since the same GSH adducts were detected in both rat and human liver microsomes. Different analytical methods could be a likely explanation. Alternatively, it is possible that this bioactivation pathway is less dominant compared to the quinine-methide pathway (Thompson et al., 1994, 1995).
Quinones are known as a class of toxicological intermediates that could cause a variety of toxic effects such as acute cytotoxicity, immunotoxicity, and carcinogenesis (Bolton et al., 2000). As highly redox active molecules, quinones can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species. As Michael receptors, quinones can alkylate cellular nucleophiles such as proteins and DNA. More recent studies have also demonstrated that some quinones and hydroquinones can poison topoisomerase II (Bender et al., 2004; Lindsey et al., 2005). Considering that 4-methyl-ortho-benzoquinone is abundant in microsomal incubations of p-cresol, the toxicological significance of its reactive metabolite should not be completely ignored. For example, as a highly redox active molecule, 4-methyl-ortho-benzoquinone might potentially play an important role in inhibition of liver mitochondria respiration caused by p-cresol (Kitagawa, 2001). It has been shown that microsomal activation of p-cresol led to oxidative DNA damage (Murata et al., 1999) and formation of DNA adducts (Gaikwad and Bodell, 2001, 2003), but the structures of formed DNA adducts have not been elucidated at present. The quinone methide generated from aliphatic oxidation of p-cresol has been proposed as the reactive metabolite leading to formation of DNA adduct (Thompson et al., 1995). Our present results raised another possibility, that, similar to quinone methide, 4-methyl-ortho-benzoquinone may also play an important role in DNA adduct formation. For instance, estrogens such as equilenin (Zhang et al., 2001) and 17ß-estradiol (Bradlow et al., 1986) formed DNA adducts via quinone intermediates. Future structural characterization of DNA adducts derived from p-cresol would provide new insights on the corresponding role of the quinone methide and ortho-quinone in covalent modification of nucleic acids.
Apparently, a quantitative comparison of the quinone methide and ortho-quinone formed in microsomal incubations would be very helpful for understanding how these two pathways may be involved in potential toxicity of p-cresol. However, such a direct comparison is not experimentally feasible due to instability of those metabolites. An alternative is to measure the level of their stable adducts. But one should realize that, since two reactive intermediates might have different reactivity with GSH, the amount of each GSH adduct formed in incubations may not necessarily be proportional to the level of their corresponding reactive metabolite. It should be noted that aldehydes may also be reactive and could covalently modify proteins and nucleic acids (Hecht et al., 2001; Kaminskas et al., 2004). The reactivity of 4-hydroxybenzylaldehyde, a major metabolite of p-cresol, remains to be determined.
In conclusion, 4-hydroxybenzaldehyde, 4-methyl-ortho-hydroquinone, and its corresponding GSH adducts were identified in incubations of p-cresol with human liver microsomes. Consequently, a new bioactivation pathway is rationalized: aromatic oxidation of p-cresol leads to the formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone. Given the fact that benzoquinones are known causative agents in the toxicity of many toxicants (Monks and Jones, 2002), the present finding is of significance in understanding the toxicology mechanism of p-cresol, and more studies are necessary in the future to further examine the possible role of 4-hydroxybenzaldehyde and 4-methyl-ortho-hydroquinone in p-cresol-induced covalent modifications of proteins and DNAs.
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Footnotes |
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ABBREVIATIONS: GSH, glutathione; P450, cytochrome P450; GSX, -glutamyl-cystein-glycin-13C2-15N; LC-MS/MS, liquid chromatographytandem mass spectrometry; MS, mass spectrometry; CID, collision-induced dissociation; NL, neutral loss; GA, glutathionyl-4-methylphenol; GB, 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone; 4HBZD, 4-hydroxybenzaldehyde; HPLC, high-performance liquid chromatography.
Address correspondence to: Zhengyin Yan, Drug Discovery, R2013, Johnson & Johnson Pharmaceutical Research & Development, LLC, Welsh & McKean Roads, Spring House, PA 19477-0779. E-mail: zyan{at}prdus.jnj.com
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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: 1,4-hydroquinone is a topoisomerase II poison. Chem Res Toxicol 18: 761–770.[CrossRef][Medline]
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