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Division of Biochemical Toxicology (Y.C., M.I.C., L.H.C., D.R.D., P.C.H.) and National Toxicology Program Center for Phototoxicology (Y.C., L.H.C., P.C.H.), National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas
(Received December 10, 2004; accepted July 8, 2005)
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Abstract |
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CYP 1B1 (no activity with CYP 2B6, 2C9, 2D6 or 2E1). The metabolism of PY74-M1 to PY74-M2 was catalyzed only by CYP 1A2 and CYP 1A1 (no activity from CYP 1B1, 2B6, 2C9, 2D6, 2E1, or 3A4). These results demonstrate that the tattoo pigment PY74 is metabolized in vitro by P450 to metabolites that should be available for phase II metabolism and excretion.
; Bäumler et al., 2000; Danish Environmental Protection Agency, 2003; Papameletiou et al., 2003) and in the United States (Cui et al., 2004).
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) or to correct skin color following some surgeries (Price et al., 2000); however, the single largest reason for voluntarily obtaining a tattoo has been personal artistic expression. In addition, permanent coloration of the lips and eyelids through tattooing is increasing in popularity and includes the application of black iron oxides and colored tattoo inks.
Tattooing has been identified as a significant risk factor for contraction of bacterial or viral (hepatitis and human immunodeficiency syndrome; HIV-AIDS) infections (Long and Rickman, 1994; Haley and Fischer, 2001; Hayes and Harkness, 2001; Hellard et al., 2004). In addition to infectious diseases, other risks associated with tattooing (reviewed in Papameletiou et al., 2003) have included acute phototoxicity, granulomatous and lichenoid reactions, development of pseudolymphomas, immunological-based rejection of the tattoo, and unwanted pigment spreading or inconsistencies within the tattoo. There have been reports of nonmelanoma skin cancer arising within tattoos (Weiner and Scher, 1987; McQuarrie, 1996; Jacob, 2002); however, these observations have not been substantiated by epidemiological studies.
The earliest tattoos were most likely derived from soot or charcoal with occasional inclusion of locally available minerals or plant products. In more recent times, inorganic compounds, such as titanium dioxide, mercuric oxide, and cadmium sulfide, have been used in tattoo inks (Lehmann and Pierchalla, 1988). The demand for an increasing number of color shades and color intensity by customers and reports of toxicity of some of the inorganic salts has led tattoo ink manufacturers to replace the inorganic salts with organic pigments to achieve the desired colors and eliminate the toxicity associated with certain metal salts. Lehmann and Pierchalla (1988) and Bäumler et al. (2000) have listed many of these pigments, which include yellow, orange, blue, green, and red pigments commonly used in the paint and commercial printing industries. Little or no toxicity data have been reported for many of these pigments, even though they contain structural alerts for mutagenicity and/or carcinogenicity.
PY74 is not listed as a hazardous substance, with an LD50 reported to be greater than 2000 to 5000 mg/kg in acute oral toxicity studies in rats, according to several Material Safety Data Sheets; however, one Material Safety Data Sheet does indicate that an irritation or allergenicity hazard exists. There have been no reports to date regarding the in vitro or in vivo metabolism or in vivo disposition of PY74. The primary objective of our study was to investigate the in vitro metabolism of PY74 by microsomal proteins and to identify the major metabolites for further evaluation of the safety of PY74 as part of our research program on tattoo ink safety.
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Materials and Methods |
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Liver microsomes from male F344 rats pretreated with 3-MC (intraperitoneal administration in corn oil for 3 consecutive days, 25 mg/kg body weight/day) were prepared by a standard procedure in our laboratory (Howard et al., 1988). The microsomal pellets were suspended in 250 mM sucrose, 50 mM potassium phosphate, pH 7.4, at approximately 20 mg/ml and stored at -80°C. Protein content was determined using the method of Lowry et al. (1951). The total cytochrome P450 (P450) content was calculated using the extinction coefficient 9.1 x 104 M-1 cm-1 from the difference spectrum of sodium dithionite-reduced and CO-saturated P450 according to the method of Johannesen and DePierre (1978). Human liver microsomes or male Sprague-Dawley rat liver microsomes from either untreated or phenobarbital (PB)-pretreated rats were purchased from In Vitro Technologies, Inc. (Baltimore, MD). Human P450 Supersomes containing expressed CYP 1A1, 1A2, 1B1, 2B6, 2C9, 2D6, 2E1, and 3A4 (with cytochrome b5) were purchased from BD Biosciences Discovery Labware (Bedford, MA). The P450 concentration (1 nmol/ml) and protein concentrations of the expressed cytochromes were based on data supplied by the manufacturer and were as follows: CYP 1A1, 45 pmol P450/mg protein; CYP 1A2, 71 pmol P450/mg protein; CYP 1B1, 91 pmol P450/mg protein; CYP 2B6, 160 pmol P450/mg protein; CYP 2C9, 179 pmol P450/mg protein; CYP 2D6, pmol P450/mg protein; CYP 2E1, 91 pmol P450/mg protein; CYP 3A4, 106 pmol P450/mg protein.
Microsomal Incubations. The PY74 stock solution was prepared immediately before the assay by mixing 0.4 ml of 1.3 mM PY74 in dimethyl sulfoxide with 10 ml of water containing 10 mg/ml bovine serum albumin. The coenzyme stock solution contained 2 mM NADP, 1.5 mM NADH, 20 mM glucose 6-phosphate, 2 units/ml glucose-6-phosphate dehydrogenase, 10 mM MgCl2, and 100 mM potassium phosphate, pH 7.4. Immediately before use, 1 mg/ml microsomal protein was added. Equal amounts (250 µl) of the two solutions were mixed at 37°C to start the reaction. Heat-inactivated microsomes (80°C for 5 min) were used in control samples. In studies investigating the dependence of PY74 metabolism on protein, PY74, or BSA concentration, the incubations were stopped at 20 min. In the time course studies, the incubations were stopped at 10, 20, 30, 45, 60, 90, and 120 min. The incubation was stopped by the addition of 3 ml of methanol/CH2Cl2 (1:2) with vigorous mixing. The CH2Cl2 was separated from the aqueous phase by centrifugation, the CH2Cl2 was collected, and the water phase was further extracted with 2 ml of CH2Cl2. After centrifugation, the organic phases were combined and 50 µl of internal standard (0.65 mM 3-nitroanisole in CH2Cl2) was added. The extract was dried using anhydrous Na2SO4 and evaporated in vacuo (SC210A Speed Vac Plus; Thermo Electron, Waltham, MA). The residue was reconstituted in 200-µl CH2Cl2 for HPLC analysis.
In the experiments to determine the specificity of human P450 isozymes in the metabolism of PY74, the incubations contained final concentrations of 0.25 nmol P450/ml, 1 mM NADP, 800 µM NADH, 10 mM glucose 6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, 4 mM MgCl2, 10 mg/ml BSA, 50 mM potassium phosphate, pH 7.4, and 25 µM PY74 (or the metabolite PY74-M1). The total volume was 200 µl and incubation time was 1 h at 37°C. The extraction and HPLC analysis were the same as described above for microsomal metabolism of PY74.
HPLC. HPLC analyses were carried out using a Waters liquid chromatography system (Waters, Milford, MA) consisting of an Alliance 2695 separation module and a 2996 photodiode array detector. The analyses were performed at ambient temperature (23°C) on a Luna C18 column (4.6 x 150 mm, 3-µm particle size) protected by a Luna C18 guard column (3 x 4 mm) (Phenomenex, Torrance, CA). The mobile phase flow rate was 800 µl/min and consisted initially of 70:15:15 water/methanol/acetonitrile, changing linearly to 5:47.5: 47.5 over 50 min, and holding for 5 additional min. UV absorbance was monitored at 254 nm and injection volumes were 20 µl (see Fig. 2).
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Mass spectra (tandem MS) were acquired on a Quattro Ultima triple quadrupole mass spectrometer (Waters, Manchester, UK) equipped with an atmospheric pressure chemical ionization (APCI) interface (heated nebulizer probe at 400°C) and an ion source temperature of 120°C. Negative ion product spectra were acquired using a collision gas cell pressure (argon) of 1.8 x 10-3 mbar and various collision energies, with a cone voltage of 40 V. The corona pin was set at 20 µA and nitrogen was used as the cone gas and desolvation gas, respectively, at 101 l/h.
Metabolite Isolation for NMR. All metabolites for NMR characterization were isolated from a 200-ml incubation using 500 µg/ml 3-MC-induced rat liver microsomal protein, 1 mM NADP, 800 µM NADH, 10 mM glucose 6-phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase, 4 mM MgCl2, 25 µM PY74, and 10 mg/ml bovine serum albumin in 50 mM potassium phosphate, pH 7.4. After 1 h, the solution was extracted with 400 ml of CH2Cl2/MeOH (2:1), and then two times with 200 ml of CH2Cl2. The organic phases were combined, dehydrated using anhydrous Na2SO4, and then concentrated to 5 to 10 ml using evaporation under reduced pressure (Rotavapor-011; Büchi Labortechnik AG, Flawil, Swizerland). The products in the extract were separated using a 4.5 x 10 cm column of 60-Å (230-400 mesh) silica gel (Merck product sold by Sigma-Aldrich). PY74 and a metabolite (PY74-M1) were eluted separately using CH2Cl2. A second metabolite (PY74-M2) was eluted from the column using a mobile phase of CH2Cl2/methanol (98:2).
The metabolite PY74-M1 was further purified using HPLC consisting of a Varian 9012 solvent delivery system (Varian Inc., Palo Alto, CA) and a Luna C18 column (4.6 x 150 mm, 3-µm particle; Phenomenex). The isocratic mobile phase consisted of water/methanol/acetonitrile (30:35:35) with a flow rate of 1.0 ml/min. UV absorbance of the eluate was monitored at 254 nm using a Varian 9050 variable wavelength UV-visible detector. The fractions containing PY74-M1 were collected and dried in vacuo.
PY74-M2 was purified using a 60-Å silica gel column (30 x 65 mm) with CH2Cl2/methanol (99:1) as the eluant. The PY74-M2 fractions were collected and dried in vacuo.
NMR Spectroscopy. Proton NMR spectra were obtained at 500 MHz on a Bruker AM-500 spectrometer (Bruker, Newark, DE). Compounds were dissolved in CD2Cl2 and all experiments were conducted at 28°C. Chemical shifts are reported in ppm by assignment of the residual methylene chloride-d1 peak (CDHCl2) to 5.32 ppm. All NMR results are from first-order measurements using either DISNMR (Bruker) or 1D WIN-NMR (Bruker).
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20 min under the same chromatographic conditions (Fig. 2), and was used in quantitative experiments.
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The metabolic activity increased significantly when the concentration of microsomal protein was increased from 100 µg/ml to 500 µg/ml, plateauing at 500 µg/ml with no additional increase in metabolism at 1 mg/ml (data not shown). The dependence of PY74 metabolism on PY74 concentration indicated a linear dependence of metabolism on the concentration of PY74, plateauing at 50 µM. Our initial intent was to conduct zero-order studies to understand the kinetics of PY74 metabolism; however, no kinetic analyses were conducted because of the complexity of the analysis (i.e., dependent on combined Kd of PY74 from BSA directly into microsomal lipid, PY74 solubility in microsomal lipid, solubility of PY74 in aqueous solution, and Km and Vmax properties of the P450).
The incubation conditions for the rat liver microsomal protein and expressed human P450s differed in the concentration of glucose 6-phosphate (10 and 5 mM, respectively) and BSA (5 and 10 mg/ml, respectively). The BSA concentration was adjusted in the human P450 studies in consideration of the low amount of P450 protein that was included in the assays. Since the studies with rat liver microsomal protein indicated that the metabolism plateaued between 5 and 10 mg/ml BSA, it was felt that the higher BSA concentration would not affect the qualitative results of the P450 assays. Similarly, since there was a low extent of P450 metabolism of both PY74 and PY74-M1, the reduced glucose 6-phosphate level in the P450 studies should not have affected the results.
The time course for the in vitro metabolism of PY74 by 3-MC-induced rat liver microsomal protein was investigated and is shown in Fig. 3. The loss of PY74 plateaued after 30 min with consumption of approximately 20% of the substrate. Since PY74-M1 and PY74-M2 had essentially the same UV-visible spectra (Fig. 2), and since we did not have a method for quantifying PY74-M1 and PY74-M2 (e.g., 3H- or 14C-labeled), we assumed that the molar extinction coefficients for PY74, PY74-M1, and PY74-M2 were equal. As a result, the HPLC eluate was monitored at 420 nm and the peak areas of all eluting compounds were quantified. The formation of PY74-M1 and PY74-M2 paralleled the loss of PY74, with a greater accumulation of PY74-M1 than PY74-M2.
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). Fractions of m/z 221 and 152 were either demethylation or deamination fragments from m/z 236 and 167, respectively. Taking into consideration the fragmentation of PY74 (Cui et al., 2004), the fragmentation of PY74-M1 is consistent with hydroxylation on the amino-anisole ring (C1''-C6'') and not on the amino-nitro-anisole ring (C1'-C6').
The NMR spectrum of PY74-M1 was obtained and compared with the previously assigned spectrum of PY74 (Cui et al., 2004). Examination of the spectrum (not shown) for PY74-M1 revealed that all proton resonances from PY74 are present with the exception of the H4'' proton at 7.13 ppm (Table 1). The resonance for proton H3'' in PY74-M1 is shifted upfield by 0.46 ppm as compared with H3'' in PY74, suggesting hydroxyl substitution at an adjacent (ortho) carbon. H3'' in PY74-M1 has only a long-range meta-coupling of 2.7 Hz to H5'', whereas H3'' in PY74 has predominantly an ortho coupling of 8.1 Hz to H4'' and meta coupling to H5''. Similarly, the resonance for proton H5'' in PY74-M1 is shifted upfield by 0.56 ppm when compared with H5'' in PY74, suggesting hydroxyl ortho-substitution. The chemical shift for H6'' in PY74-M1 is 0.19 ppm upfield from H6'' in PY74, indicating that the 6-position is meta to the site of substitution on the ring. The resonances for H6'' and H5'' in PY74-M1show ortho coupling constants (8.7 Hz). Taking into account these observations, we conclude that the site of hydroxylation on PY74-M1 is the C4''-position as shown in Fig. 4A. The appearance of a substantially broadened resonance at 4.94 ppm (Table 1) is attributable to the 4''-OH. The combined LC/MS and NMR analysis support that the structure of PY74-M1 is 2-((2-methoxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxo-butanamide.
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The addition of PY74-M1 to microsomal incubations containing 3-MC-induced rat liver microsomes indicated that PY74-M1 is metabolized to PY74-M2 (data not shown). The APCI negative-ion MS analysis of purified PY74-M2 is shown in Fig. 4B and indicates a parent ion [(M - H)-] at m/z 387, which is a loss of 14 Da from PY74-M1. The most probable structure of PY74-M2 to account for this mass is a demethylation product of PY74-M1. Fragmentation ions at m/z 152 and m/z 222 (Fig. 4B) suggest that the site of demethylation is the methoxy group on the amino-nitro-anisole ring (C1'-C6') and not the amino-anisole ring (C1''-C6'').
The proton NMR spectrum of PY74-M2 confirmed the site of demethylation. PY74-M2 had the same number of proton resonances and a coupling pattern similar to that of PY74-M1, with the one notable exception that there was only a single methoxy resonance (integrated to approximately 3 hydrogens) at 3.93 ppm, indicating a loss of one of the methoxy groups (Table 1). The chemical shift of this methoxy group (3.93 ppm) was the same as that of the C7'' methoxy in PY74-M1, which was only shifted 0.04 ppm upfield from the C7'' group in PY74 (Table 1). The resonance of the methoxy group at C7' (4.08 ppm in PY74-M1 and 4.09 ppm in PY74) was not present in PY74-M2. This finding suggested that PY74-M2 was the C7' O-demethylation product of PY74-M1. This assignment is further supported by a 0.29-ppm upfield shift for the H6' proton, which is meta to the C7' hydroxyl. Thus, the structure of PY74-M2 was identified as 2-((2-hydroxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxo-butanamide. This metabolite is not stable in water at room temperature and decomposes to more polar compounds.
Metabolism of PY74 and PY74-M1 by Human Cytochromes P450. The incubation of PY74 with human liver microsomes (not shown) resulted in the formation of predominantly PY74-M1 and PY74-M2, with one minor peak that was less polar than PY74-M1, which was not identified due to lack of sufficient material. The specificity of the human P450 metabolism of PY74 is shown in Fig. 5, where CYP 1A2 had the highest activity, followed by CYP 1A1, 3A4, and 1B1. CYP 2B6, 2C9, 2D6, and 2E1 did not metabolize PY74. CYP 1A1 and CYP 1A2 metabolized PY74 to both PY74-M1 and PY74-M2 at a ratio of approximately 5.6:1, whereas CYP 1B1 formed exclusively PY74-M1 (data not shown).
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; Bäumler et al., 2000; Cui et al., 2004). To date, neither the metabolism of PY74 nor the description of the identity of any metabolites of PY74 has been presented.
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We find that PY74 is metabolized by microsomal protein from 3-MC-induced rat livers. 3-MC is an inducer of CYP1A proteins (Tanaka et al., 1997). We found that the PY74 metabolism by microsomal proteins from noninduced or phenobarbital-induced rat livers was considerably less than that from the 3-MC-induced livers. This is consistent with our observations that CYP 1A1 and 1A2 were the isoforms responsible for PY74 metabolism because phenobarbital is an inducer primarily of CYP 2Bs and, to some extent, 3As (Martignoni et al., 2004). These data are consistent with the human P450-specificity of PY74 metabolism, where CYP 1A1 and CYP 1A2 had the highest activity of the tested P450s for PY74 metabolism (Fig. 5). We found that PY74-M1 is also preferentially metabolized by CYP 1A1 and 1A2 to its O-demethylated product (Fig. 6). These data suggest that tissue expressing CYP 1A1 or CYP 1A2 would have the capacity to metabolize PY74.
The skin is an important organ in the human body, not only serving as a barrier to the environment, but also having the capacity to metabolize chemicals that enter the body by absorption through the skin. The skin does contain P450s, albeit at low levels compared with other organs such as the liver and kidney (Mukhtar and Khan, 1989). The P450s in skin can be induced severalfold by a wide variety of substrates (Mukhtar and Bickers, 1981; Mukhtar and Khan, 1989) and are altered by exposure to ultraviolet light (Goerz et al., 1996). Janmohamed et al. (2001) have reported that mRNAs for CYP 2A6, 2B6, and 3A4 are expressed in human skin taken from surgical samples and that these P450s were primarily located in the epidermis, sebaceous glands, and hair follicles. Yengi et al. (2003) reported the presence of mRNA for CYP 1A1, 1B1, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5 in human skin, whereas CYP 1A2, 2A6, and 2C8 mRNA levels were below the detection limits of their assay. Ahmad et al. (1996) reported that CYP 1A1 was the major P450 in rodent skin, which is consistent with reports of the induction of P450 activity in the skin of rodents treated with 3-MC and other inducers of CYP 1A. Du et al. (2004) reported that 13 cytochromes of the CYP2 family, including 2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1, were present in human skin samples. In summary, the skin contains many different P450s, which have been detected as either mRNA, enzyme activities, or protein levels.
Our work has shown that PY74 is metabolized by human CYP 1A1, 1A2, 1B1, and 3A4. These P450s are present in the skin of rodents and humans, suggesting that metabolism of PY74 could occur there in vivo.
We have identified the major metabolite of PY74 as the 4''-hydroxylation product PY74-M1 through both MS and NMR techniques. Our conclusion that PY74 is hydroxylated at the 4''-position is consistent with the work of Sapota et al. (2003), who demonstrated that administration of 2-methoxyaniline (o-anisidine) to rats results in the excretion of N-acetyl-2-methoxyaniline and N-acetyl-4-hydroxy-2-methoxyaniline. o-Anisidine and its N-acetyl derivative are structurally very similar to the C1''-C6'' ring system on PY74.
The second metabolite is an O-demethylation of PY74-M1 by CYP1A1 and 1A2. Many P450s are capable of O-demethylation of xenobiotics. For example, CYP 2D6 catalyzes the O-demethylation of dextromethorphan, hydrocodone, 5-methoxy-N,N-dimethyltryptamine, and pinoline.
We believe our results demonstrate that PY74-M1 is sequentially converted to PY74-M2. First, the incubation of PY74-M1 with either microsomal protein or P450 resulted in the formation of PY74-M2 (Fig. 6). Second, the structures of PY74-M1 and PY74-M2 are consistent with sequential metabolism. Third, additional metabolites such as the 7'-O-demethylation of PY74, which would be expected as a precursor to PY74-M2, were not detected. If labeled (3H, 13C, or 14C) PY74 becomes available, future experiments would be able to confirm the proposed sequential metabolism of PY74 to PY74-M1 and then PY74-M1 to PY74-M2.
PY74 is a nitro-aromatic compound that contains an azo (or hydrazone; Cui et al., 2004) group. Many azobenzenes have been reported to be reduced to primary amines by P450s and NADPH-cytochrome P450 reductase (Levine, 1991). Metabolic azo reduction and cleavage is believed to be an activation reaction since the reduction products of some azo compounds exhibit toxic and mutagenic effects (Chung, 1983; Martin and Kennelly, 1985). A structure-activity study of various azobenzenes revealed that a polar electron-donating group (amino or hydroxyl) para to the azo linkage is required for substrate activity with microsomes. An electron-withdrawing group, such as a nitro group, on the benzoyl moiety prevents electron donation to the azo ring system, and the resulting product is inactive in azo reduction (Zbaida, 1995). Although we did not examine the azo reduction of PY74 by skin microsomes or cytosol in our metabolic system, skin azo reduction of several azo compounds including 5-(phenylazo)-6-hydroxynaphthalene-2-sulfonic acid, Sudan I (1-phenylazo-2-naphthol; C.I. 12055), and Solvent Yellow 7 (4-phenylazophenol; C.I. 11800) has been demonstrated with a percutaneous absorption system (Collier et al., 1993). Therefore, the potential for azo reduction of PY74 by skin does exist and should be investigated. In addition, simulated solar light (Cui et al., 2004) and laser light used for tattoo removal (Vasold et al., 2004) have been shown to photochemically cleave PY74 at the azo group to generate aromatic amines, such as 2-methoxy-4-nitroaniline and 2-aminoanisole from PY74.
Under the definitions given in the Food Drug and Cosmetic Act, and associated regulations in 21 US Code of Federal Regulations Title 21, tattoo inks have been categorized by the Food and Drug Administration (FDA) to be professional use cosmetics. As a result, the pigments contained in the inks are considered to be color additives. Both cosmetics and color additives fall under the regulatory authority of the FDA. Although individuals are encouraged to report adverse events resulting from FDA-regulated products to the U.S. Food and Drug Administration, the lack of public perception that tattoo inks are regulated by the FDA probably explains why few complaints have been received, thus explaining why a comprehensive assessment or database on adverse events resulting from tattooing does not exist.
The results described in this paper show that tattoo ink pigments can be metabolized by phase I enzymes (P450s) that exist in the skin of rodents and humans. Anecdotal reports of tattoo fading are routinely heard from tattoo recipients who have yellow and orange tattoos. We suggest that the mechanisms for fading could include: 1) dispersion through the skin; 2) phagocytosis and removal; 3) metabolism of the pigments in the skin (this report); or 4) photochemical decomposition of the pigments as reported for PY74 (Cui et al., 2004; Vasold et al., 2004). No reports exist regarding the mutagenic, carcinogenic, or photocarcinogenic potential of PY74; however, 2-amino-anisole (o-anisidine) can be enzymatically activated in vitro to form DNA adducts (Stiborova et al., 2001, 2002) and is a urinary bladder carcinogen in mice and rats (National Toxicology Program, 1978). Oxidation of PY74-M1 could result in the formation of structure similar to a p-quinone imine, which is the active intermediate for o-anisidine (Stiborova et al., 2002) and acetaminophen (N-acetyl-p-benzoquinone imine; Rogers et al., 1997). To conduct a risk assessment for PY74, further studies on the purity of PY74 used in tattoo inks, the in vivo metabolism of PY74 in the skin, and quantification of the toxicity of its metabolites are among some of the studies that are warranted.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: PY74, Pigment Yellow 74; PY74-M1, metabolite 1 of PY74; PY74-M2, metabolite 2 of PY74; BSA, bovine serum albumin; 3-MC, 3-methylcholanthrene; HPLC, high performance liquid chromatography; P450, cytochrome P450; PB, phenobarbital; MS, mass spectrometry; APCI, atmospheric pressure chemical ionization; FDA, Food and Drug Administration.
Address correspondence to: Dr. Paul C. Howard, Division of Biochemical Toxicology, HFT-110, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079. E-mail: Phoward{at}nctr.fda.gov
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drzejczak M (2003) Tissue distribution, excretion and metabolism of o-anisidine in rats. Int J Occup Med Environ Health 16: 351-357.[Medline]
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) values are in ppm, and coupling constant (J) values are in Hz. Splitting patterns: s, singlet; bs, broad singlet; d, doublet; dd, double of doublets; dt, double of triplets.
