Nicotine and Cotinine Adducts of a Melanin Intermediate

Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 l-DOPA:drug ratio. A black precipit...
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Chem. Res. Toxicol. 2001, 14, 275-279

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Nicotine and Cotinine Adducts of a Melanin Intermediate Demonstrated by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Donna L. Dehn,† David J. Claffey,† Mark W. Duncan,‡ and James A. Ruth*,† Program in Molecular and Environmental Toxicology and Biochemical Mass Spectrometry Facility, School of Pharmacy, The University of Colorado Health Sciences Center, Denver, Colorado 80262 Received September 14, 2000

Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis.

Introduction Drug testing employing hair as an alternative specimen to urine, plasma, or blood is becoming widespread (1). The chemical mechanisms underlying drug accumulation and retention in hair have not been elucidated; however, numerous investigators have observed that hair pigmentation is a determining factor in the extent of incorporation of several drugs into the hair matrix (26). Drug-melanin interactions have been reported for codeine (7), methadone (8), cocaine (9, 10), and buprenorphine (3) to name just a few. In addition, autoradiographic studies carried out in our laboratory have demonstrated that within minutes of systemic administration there is accumulation of radiolabeled nicotine, cocaine, and flunitrazepam over melanin granules in developing hair follicles in eumelanin-pigmented mice (11). Melanin is formed within the melanosome of melanocytes through the oxidative action of the enzyme tyrosinase on tyrosine. Tyrosine is oxidized to DOPA and then to dopaquinone (DOPAQ) with subsequent ring closure and formation of an indole, dopachrome. Dopachrome undergoes rearrangement to form 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (12). Although the general scheme of melanogenesis was outlined in 1927 by Raper (13), many aspects of the structure have been elusive due to the physical and chemical properties of melanin (i.e., largely insoluble, * To whom correspondence should be addressed: 4200 E. Ninth Ave., Box C-238, Denver, CO 80262. Phone: (303) 315-7569. Fax: (303) 3156281. E-mail: [email protected]. † Program in Molecular and Environmental Toxicology. ‡ Biochemical Mass Spectrometry Facility.

highly inhomogeneous, tightly associated with other cellular components, significant alteration upon degradation, and lacking well-defined spectral characteristics) (14). Recent studies have utilized MALDI-TOF MS1 (matrixassisted laser desorption/ionization time-of-flight mass spectrometry) in the investigation of the structure of melanin (14-16). These studies have demonstrated that melanin is composed of oligomers of DHI and DHICA together with further oxidation products. The poly(oquinone) nature of melanins and the oxidative environment in which they are formed provide several modes of adduct formation with a variety of chemical functionalities. Lyden et al. (17) speculate that the free carboxyl groups of DHICA bind the protonated amino groups of basic drugs in a purely electrostatic interaction. However, we have demonstrated that radiolabeled flunitrazepam and nicotine accumulated in the hair of pigmented mice could not be completely removed, even in the face of vigorous degradative treatments (11, 18). These data indicate a much stronger drug-melanin interaction than one based on ion pairing alone. In fact, Palumbo et al. (19) have reported a covalent interaction of 2-thiouracil, an antithyroid drug, with dopaquinone. Further, Manini et al. (20) recently reported a L-cysteine-dopaquinone adduct formed as a urinary metabolite. The purpose of this study was to test the hypothesis that drug adducts could be observed in the tyrosinasemediated formation of eumelanins upon inclusion of 1 Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/ ionization mass spectrometry; DHI, dihydroxyindole; DHICA, 5,6dihydroxyindole-2-carboxylic acid; CHCA, R-cyano-4-hydroxycinnamic acid; DOPAQ, dopaquinone.

10.1021/tx000205l CCC: $20.00 © 2001 American Chemical Society Published on Web 02/01/2001

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nicotine or cotinine in the reaction mixture. Nicotine was employed because of its strong association with melanin (11, 18). To test this hypothesis, melanin was synthesized in vitro in the presence of nicotine, cotinine, and their deuterated analogues. The resulting mixture was examined by MALDI-TOF MS. This approach allows ionization, ion separation, and detection of nonvolatile compounds without significant fragmentation. This technique has been employed by other investigators to confirm the presence of adducts to proteins and DNA (21). MALDITOF MS provides accurate mass assignments and can also deliver additional structural information about masses of interest when postsource decay of the parent ion is performed.

Methods L-3,4-Dihydroxyphenylalanine (L-DOPA),

tyrosinase (mushroom), and (-)-nicotine were purchased from Sigma Chemical Co. (St. Louis, MO). L-DOPA-d3 (ring-d3, 98%) was purchased from Cambridge Isotope Laboratories (Andover, MA). DL-Nicotined3 (methyl-d3) and (S)-(-)-nicotine-2,4,5,6-d4 (pyridine-d4) were obtained from Isotec, Inc. (Miamisburg, OH). Cotinine and cotinine-d3 (methyl-d3) were obtained from Radian International (Austin, TX). Water was deionized and had a resistivity of 20 MΩ. Melanin was synthesized in vitro as described by Bertazzo et al. (15). Here, L-DOPA (1 mg/mL solution) was treated with mushroom tyrosinase (1000 units/mL) in water at 37 °C. Additional sample sets were prepared under the same conditions except either (-)-nicotine, DL-nicotine-d3, (S)-(-)-nicotine-2,4,5,6d4, cotinine, or cotinine-d3 at a 10:1 L-DOPA:drug ratio was added at the beginning of the oligomerization reaction. A black precipitate formed within 60 min. Aliquots (10 µL) were removed from the incubation mixture at 60, 120, and 360 min. These were analyzed directly because previous studies (unpublished data) indicate that centrifugation and/or filtration was unnecessary. Alternatively, melanin was synthesized in the absence of enzyme by heating L-DOPA (1 mg/mL aqueous solution) in a capped vial overnight at 37 °C. Sample sets were also prepared with and without the drugs listed above. After 24 h, a black precipitate had formed. The samples were mixed thoroughly, and aliquots (10 µL) were taken for analysis. An additional sample set was prepared by substituting L-DOPA-d3 for L-DOPA both with and without enzyme and with nicotine or one of its deuterated analogues. Samples with enzyme added exhibited an immediate color change (dark orange), and a black precipitate formed within 60 min. Sample sets without enzyme required 24 h at 37 °C before the black precipitate formed. An aliquot (10 µL) was taken from each sample after mixing. Each 10 µL sample aliquot was mixed 1:2 (v/v) with the matrix solution. The matrix was prepared daily by dissolving 10 g of freshly recrystallized R-cyano-4-hydroxycinnamic acid (CHCA, Sigma Chemical Co.) in 1 mL of a 70:30 acetonitrile/ water mixture containing 0.1% trifluoroacetic acid. A 1 µL aliquot of the sample/matrix mixture was applied to a stainless steel MALDI target and was allowed to crystallize at room temperature and pressure. A PerSeptive Biosystems Voyager-DE PRO time-of-flight mass spectrometer (Applied Biosystems Inc., Foster City, CA) equipped with delayed extraction and a nitrogen laser (337 nm, with a focal diameter of 25 µm) was used for all analyses. The flight tube length in the reflector mode is 2 m. Spectra were acquired in the positive ion reflectron mode using an accelerating voltage of 20 kV. The reflectron mode was used because the increased resolution aids in separating low-molecular mass sample ions from matrix ions and increases mass accuracy. Mass spectra were analyzed over the range of m/z 100-2500 by averaging the data from 64 laser shots. Multiple mass determinations (n ) 4-9) were made on each sample to provide a

Figure 1. Representative MALDI-TOF spectrum of melanin oligomers formed in vitro after incubation (1 h, 37 °C, and pH 7) of (A) L-DOPA (1 mg/mL) or (B) L-DOPA-d3 (1 mg/mL) and mushroom tyrosinase (1000 activity units) in water. The internal calibration ion, m/z 379.0930, is shown in bold type. mean and standard error for each of the masses of interest. Internal calibration across the mass range was performed and employed the [M + H]+ ions of CHCA (m/z 190.0504 and 379.0930). These bracketed the analyte ions of interest. The instrument resolution in this mass range was 18 000. Postsource decay (PSD) fragment ion analysis was performed on masses of interest by ion gating. Approximately 100 spectra were accumulated per PSD ion. PSD fragments were recorded by stepping the reflectron voltage in 12 steps and stitching the individual scans.

Results The spectra from all samples (either L-DOPA or its deuterated analogue, with and without enzyme, and with and without drug) were compared by plotting the mass of each ion that was at least 3-fold greater than the baseline noise intensity on a scatter plot diagram. Spectral data from each of the replicates of a single sample were reviewed, and all ions meeting the stated criteria were included in the plot of a specific sample. Matrix ion masses were not included on the diagrams. This allowed the identification of masses common between samples and unique to a sample. Figure 1A is the spectrum of L-DOPA after a 1 h incubation with tyrosinase (37 °C and pH 7). Although an immediate dark orange color change occurred, ions corresponding to the oligomerization of DHI centered around m/z 296 (dimer), 441 (trimer), 588 (tetramer), and 736 (pentamer) were not evident until a precipitate formed at ∼1 h. Note that ions at m/z 441, 558, and 736 display a fine structure that is not characteristic of such low-mass ions. Each central group of oligomer peaks is

MALDI-TOF MS of Nicotine-Melanin Adducts

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Figure 3. Representative MALDI-TOF spectrum of a reaction mixture formed in vitro after incubation (1 h, 37 °C, and pH 7) of L-DOPA-d3 (1 mg/mL), nicotine (0.1 mg/mL), and mushroom tyrosinase (1000 activity units) in water. The internal calibration ion, m/z 379.0930, is shown in bold type.

Figure 2. Representative MALDI-TOF spectrum of a reaction mixture formed in vitro after incubation (1 h, 37 °C, and pH 7) of L-DOPA (1 mg/mL) and (A) nicotine (0.1 mg/mL), (bold type showing the m/z 358.1767 ion-calculated mass from eight separate samples), (B) nicotine-d3 (0.1 mg/mL) (asterisk indicating that this peak is representative of all spectra that were obtained), or (C) nicotine-d4 (0.1 mg/mL) (pound sign indicating that this peak is representative of all spectra that were obtained) and mushroom tyrosinase (1000 activity units) in water. The internal calibration ion, m/z 379.0930, is shown in bold type.

also flanked on both the high and low mass side by a less intense cluster of peaks 16 ( 2 Da away. Substituting L-DOPA-d3 results in the spectrum (at 1 h) shown in Figure 1B. Deuterated DOPA had no effect on the timing of melanin formation, as was seen by a rapid change in the solution color. The major oligomer peaks shifted to m/z 300, 447, 596, and 744. The dimer at m/z 296 [M + H]+ in panel A shifts to 300 in panel B due to the presence of four deuterons. A similar pattern of groups of peaks 16 ( 2 Da on either side of the major group of peaks is again evident. Panels A-C of Figure 2 show representative MALDITOF MS spectra of a reaction mixture formed in vitro after a 1 h incubation of L-DOPA (1 mg/mL, 37 °C, and pH 7) and (A) nicotine (0.1 mg/mL), (B) nicotine-d3 (0.1 mg/mL), or (C) nicotine-d4 (0.1 mg/mL) and mushroom tyrosinase (1000 activity units) in water. The ion at m/z 358.1767 (n ) 9, SE ) 0.002) corresponds to the covalent adduct of nicotine and DOPAQ. The use of deuterated nicotine confirms that the nicotine nucleus is associated with the compound at m/z 358.1767 because the mass shifts to m/z 361.1776 (n ) 4, SE ) 0.002) with nicotine-d3 and to m/z 362.1661 (n ) 5, SE ) 0.022) with nicotine-d4. Figure 3 shows a representative MALDI-TOF MS spectrum of the protonated DOPAQ monomer-nicotine adduct formed in vitro after a 1 h incubation of L-DOPA-

d3 (1 mg/mL, 37 °C, and pH 7) and nicotine (0.1 mg/mL) with mushroom tyrosinase (1000 activity units) in water. The use of L-DOPA-d3 confirms that this nucleus is part of the structure giving rise to the ion at m/z 358.1767 (shown in Figure 2A) because the peak shifts to m/z 360.1878 (n ) 5, SE ) 0.005) as a result of incorporation of the isotope. The loss of one ring deuteron occurs upon substitution of nicotine at a ring carbon atom of DOPAQ. To determine whether adduct formation extended to nicotine metabolites, cotinine and cotinine-d3 were employed in the same strategy. Panels A and B of Figure 4 show the MALDI-TOF MS spectra of the reaction mixture formed after a 1 h incubation of L-DOPA (1 mg/mL, 37 °C, and pH 7) with cotinine (0.1 mg/mL) and cotinine-d3 (0.1 mg/mL), respectively, with mushroom tyrosinase (1000 activity units) in water. The ion at m/z 372.1649 (n ) 5, SE ) 0.004) corresponds to cotinine covalently bound to DOPAQ. A shift to m/z 374.9262 (n ) 6, SE ) 0.051) with cotinine-d3 confirms the presence of drug within that specific molecular mass. On the basis of mass, spontaneous oligomerization (no enzyme added to L-DOPA solution) results in the formation of the same masses corresponding to DOPAQ and adduct peaks (data not shown), but at a slower rate. No initial color change occurred, and 24 h was required before the solution turned dark brown and formed a precipitate. The proposed structures for the DOPAQ-nicotine and -cotinine adducts are shown in Figure 5. The calculated mass value for the DOPAQ-nicotine adduct ([M + H]+ m/z 358.1767, n ) 9) was not significantly different from the theoretical mass of the proposed product (i.e., a mass difference of 0.2 ppm). The mass accuracy for the cotinine adduct ([M + H]+ m/z 372.1649, n ) 5) is 24 ppm. The fragment ions from PSD analysis of the m/z 358.1767 ion are shown in Figure 6. Major fragments were observed at m/z 301 (loss of methylaminoethylene), 275 (loss of pyrroline), 227 (loss of formic acid and hydrogen), and 200 (loss of glycine).

Discussion In the course of investigating mechanisms of drug incorporation into hair, we have observed that interactions between administered drugs and some endogenous serum constituents with melanin in hair appear to be partially irreversible (11, 18, 22-24). It is possible that

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Figure 6. PSD spectrum of the nicotine-DOPAQ adduct with sources of the observed fragment ions.

Figure 4. Representative MALDI-TOF spectrum of a reaction mixture formed in vitro after incubation (1 h, 37 °C, and pH 7) of L-DOPA (1 mg/mL) and (A) cotinine (0.1 mg/mL) and (B) cotinine-d3 (0.1 mg/mL) and mushroom tyrosinase (1000 activity units) in water. The internal calibration ion, m/z 379.0930, is shown in bold type.

Figure 5. Proposed structures of DOPAQ-nicotine and -cotinine adducts with the observed and calculated mass values.

the apparent irreversibility observed in in vivo studies is a function of structural barriers in the hair matrix, or in the membranes of the melanosomes in desiccated keratinocytes of the hair shaft preventing release of radiolabeled drugs associated with melanin (18). However, recent in vitro polymerization studies indicate that drug-melanin association is chemical, perhaps covalent in nature.2 The current study was undertaken in an effort to provide direct evidence of a drug-melanin association. Polymerization of L-DOPA by tyrosinase (the system which gives rise to eumelanin in vivo) in the presence of nicotine or cotinine results in products with mass values consistent with the proposed structures of the adducts. The polymerization of L-DOPA-d3 in the absence of drug demonstrates that the DHICA and DHI oligomer peaks identified are in fact derived from L-DOPA. The mass 2

D. J. Claffey et al., manuscript submitted for publication.

shifts in the proposed adduct peaks obtained with nicotine-d3, nicotine-d4, and cotinine-d3 further support the formation of the proposed structures. The fine structure (clusters) of the melanin oligomer ions is consistent with the presence of several oxidized and reduced forms of the same species being present in the reaction mixture, and is not typical of low-molecular mass ions. While it is likely that these are constituents of the reaction mixtures, we cannot exclude the possibility that they were formed in part in the MALDI-TOF instrument when the sample was irradiated. Attempts to generate adducts with L-DOPA-d3 and isotopically labeled nicotine and cotinine did not reproducibly afford products with mass shifts of 5 and 6 ppm. The reason for this is not known, but may involve deuterium isotope effects which preclude the formation of the d5- and d6-adducts. The adducts have tentatively been assigned the structures shown in Figure 5, which result from addition of the 4-pyridinyl radical of nicotine or cotinine to the 6-position of DOPAQ. Analogous adducts have been reported with 2-thiouracil and DOPAQ (19), and with cysteine (20). A hypothetical mechanism for the formation of the nicotine and cotinine adducts with DOPA is outlined in Scheme 1. L-DOPA (1) is oxidized by tyrosinase to DOPAQ (2). Nicotine (3) (or cotinine) undergoes a oneelectron transfer from cuprous ion in tyrosinase to afford the 1,4-radical anion 4. Other sources are available for this reduction, such as coupling to DOPA oxidation (25). Addition of the 4-pyridinyl radical to DOPAQ would give rise to the radical 5. A 1,3-hydrogen shift to the quinone radical to generate the quinone 6 is supported by the observations from polymerization with pyridine ringlabeled nicotine-d4, where the adduct mass is shifted by 4 mass units. This is possible only if one deuterium atom of the nicotine is transferred to a nonexchangeable site on the quinone residue. This hydrogen shift would be accompanied by aromatization of the pyridine ring. The resulting quinone 6 is a tautomer of 7. In the tautomer-

MALDI-TOF MS of Nicotine-Melanin Adducts Scheme 1

ism of deuterio 6, the C-D bond would be expected to be retained on the basis of the lower resting energy of the C-D bond versus the C-H bond (26). Examination of the reaction mixtures for adducts of nicotine or cotinine to higher-molecular mass melanin oligomers has not yet been completed because of the complexity of the mixture. The postsource decay examination of the m/z 358 ion of the nicotine adduct demonstrates fragments consistent with the structure of the proposed adduct. The pyrrolidine ring of the nicotine portion of the structure is lost, as is glycine, or fragments thereof, from the quinone portion of the adduct. A difference in mass from theoretical of only 0.2 ppm further supports the assignment. The mass accuracy of the cotinine adduct (24 ppm), although higher than that generally obtained in the reflectron mode, is still supportive of the suggested structure. However, definitive structural assignment is contingent upon unambiguous synthesis. The report of characterization of this adduct is important, however, since it is the first reported adduct of a melanin precursor and a widely employed drug of significance in hair analysis. The potential value of drug-melanin adducts lies in their characterization for possible use as an indicator of drug ingestion.

Acknowledgment. This work was supported by U.S. Public Health Service Grant DA09545. We are indebted to Drs. Franco Basile and Joseph Zirrolli of the Biochemical Mass Spectrometry Facility for discussions and their skill in obtaining the PSD spectra.

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