1420
Chem. Res. Toxicol. 1997, 10, 1420-1426
Amination of Tyrosine in Liver Cytosol Protein of Male F344 Rats Treated with 2-Nitropropane, 2-Nitrobutane, 3-Nitropentane, or Acetoxime Rama S. Sodum* and Emerich S. Fiala Division of Biochemical Pharmacology, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received August 5, 1997X
Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.11.5 mol of 3-aminotyrosine/103 mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at ∼0.01 mol/103 mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-Osulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-Osulfonates as intermediates.
Introduction 2-Nitropropane, a component of cigarette smoke (1) and an important industrial chemical (2), is a mutagen in bacteria (3, 4), produces unscheduled DNA synthesis in rat hepatocytes (5-7), and is a hepatocarcinogen in rats by inhalation or gavage (8, 9). 2-Nitropropane and related secondary nitroalkane carcinogens such as 2-nitrobutane and 3-nitropentane (7) produce characteristic base modifications in rat liver RNA and DNA, including the amination and the oxidation of C8 of guanine. Both of these modifications are detectable using HPLC with electrochemical detection (10, 11). By using in vitro and in vivo methods, we demonstrated that the production of liver nucleic acid modifications by secondary nitroalkanes depended on their activation by aryl sulfotransferase (7, 12). In contrast to secondary nitroalkanes, the primary nitroalkanes 1-nitropropane and 1-nitrobutane are not mutagenic (4) or carcinogenic in rats (7, 9), do not serve as substrates of aryl sulfotransferase in vitro, and also do not produce nucleoside modifications discernible by HPLC with electrochemical detection (7, 10). The mechanism we proposed (11, 12) for the sulfotransferasecatalyzed activation of 2-nitropropane and related secondary nitroalkanes is shown in Scheme 1. It involves ionization of the secondary nitroalkane (I) to the nitronate ion (II) followed by esterification either of the * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, November 15, 1997.
S0893-228x(97)00137-9 CCC: $14.00
nitronate or, less probably, of the nitronic acid (III) with sulfate and reduction to the corresponding oxime-Osulfonate (IV). The oxime-O-sulfonate can then hydrolyze nonenzymatically to hydroxylamine-O-sulfonate (VI), which yields a reactive aminating intermediate, most probably the unsubstituted nitrenium ion, NH2+ (VII) (13). The primary nitroalkanes examined thus far do not undergo this activation pathway and cannot produce the nucleic acid modifications that are characteristic of the secondary nitroalkanes because they lack a sufficiently acidic R hydrogen (14) and cannot therefore undergo sulfation at physiological pH. In this report we present additional findings in support of the mechanism we proposed for the activation of 2-nitropropane and other secondary nitroalkanes. We demonstrate that 2-nitropropane, other secondary nitroalkanes, and also acetoxime (which has been postulated to be metabolically oxidized to 2-nitropropane nitronate; see refs 15-17) produce a novel protein modification in vivo, i.e., the amination of tyrosine to 3-aminotyrosine. As expected, the primary nitroalkanes are inactive in this respect. In addition, we show that acetoxime-O-sulfonate and hydroxylamine-O-sulfonate, intermediates in the proposed mechanism in Scheme 1, aminate not only guanosine but also tyrosine in vitro.
Experimental Procedures Caution: 2-Nitropropane, 2-nitrobutane, 3-nitropentane, and acetoxime are carcinogens in rodents and should be handled with care and appropriate protection.
© 1997 American Chemical Society
Tyrosine Amination by Nitroalkanes and Acetoxime
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1421 Scheme 1
Materials. Acetoxime, 2-nitropropane, 2-nitrobutane, 3-nitropentane, 1-nitrobutane, 1-nitropentane, acetophenone, 2-heptanone, L-tyrosine, 3-hydroxy-L-tyrosine, and 1-nitropropane were purchased from Aldrich Chemical Co. (Milwaukee, WI). 3-Amino-L-tyrosine, 8-aminoguanosine, bovine serum albumin, alkaline phosphatase, and nuclease P1 were obtained from Sigma Chemical Co. (St. Louis, MO). 8-Oxoguanosine and N1aminoguanosine were prepared according to published procedures (18, 19). Constant-boiling 6 N HCl and protein hydrolysis tubes were obtained from Pierce Chemical Co. (Rockford, IL). Nucleosil C18 HPLC columns and guard columns were purchased from Phenomenex (Torrance, CA), and Whatman Partisphere SCX HPLC columns and guard columns were obtained from Scientific Products (Edison, NJ). Ultrasphere ODS columns were purchased from Alltech (Avondale, PA). In all cases particle size of the HPLC packings was 5 µm. Animals. The nitroalkanes or acetoxime was dissolved in an aqueous 10% solution of Emulphor 620 (GAF Corp., Wayne, NJ) for administration to rats. 2-Nitropropane, 2-nitrobutane, 3-nitropentane, and 1-nitropropane were administered to male F344 rats (Taconic Labs, Germantown, NY) by ip injection once, at a dose of 1 mmol/kg of body wt. Acetoxime, 3.3 mmol/kg of body wt, was administered once by the same route. Rats were sacrificed 6, 18, or 23 h after administration, and the livers were removed and frozen immediately in liquid nitrogen before storage at -80 °C. HPLC Instrumentation. Solvent was delivered with the Shimadzu model LC-600 HPLC pumps (Shimadzu Scientific Instruments, Braintree, MA) with Shimadzu model SCL-6B solvent programmer. The eluate was analyzed using the Waters model 996 photodiode array detector and the BAS (Bioanalytical Systems, West Lafaytte, IN) model LC-4B amperometric detector with a glassy carbon working electrode and the Ag/AgCl/3 M NaCl reference electrode. An electrode potential of 475 mV was used for analysis of protein hydrolysates and 600 mV was used for nucleoside analysis. Data were collected and processed using the Waters Millennium Chromatography Manager. HPLC Systems. System 1: cation-exchange HPLC using a Whatman Partisphere SCX column (4.6 × 125 mm) connected to a Partisphere SCX guard column, eluted with 25 mM sodium citrate with 6% methanol, pH 3.2, at a flow rate of 0.7 mL/min. System 2: ion-pair chromatography using a Nucleosil C18 (4.6 × 250 mm) reverse-phase column with a Nucleosil C18 guard column (4.6 × 30 mm), eluted with 25 mM sodium citrate containing 5 mM heptanesulfonic acid and 10% methanol, pH
3.3, at a flow rate of 1 mL/min. System 3: reverse-phase HPLC using a Nucleosil C18 (4.6 × 250 mm) reverse-phase column with a Nucleosil C18 guard column, eluted isocratically with 25 mM sodium acetate and 12.5 mM sodium citrate containing 5% methanol, pH 4.9, at a flow rate of 1.0 mL/min. Protein Sample Preparation. Rat livers were thawed at 4 °C and homogenized, and the cytosol supernatant was obtained by centrifugation at 107000g. Protein concentrations in the cytosolic fractions were ∼25 mg/mL as determined using the Sigma protein determination kit. Protein was obtained from the cytosolic fractions by precipitation with excess acetone containing 0.2% HCl and was then dried under nitrogen. The protein was hydrolyzed under vacuum in protein hydrolysis tubes with 6 N HCl at 150 °C for 1 h; then the HCl was removed by evaporation under a stream of nitrogen. Under these hydrolysis conditions 85% of added 3-aminotyrosine could be recovered. The dry residue was redissolved in 1-2 mM sodium citrate, pH 3.5, and analyzed by ion-pair and cation-exchange HPLC with UV and EC detectors. Reaction of Guanosine with Hydroxylamine-O-sulfonic Acid and Acetoxime-O-sulfonate. Reactions of guanosine (10 mM) with hydroxylamine-O-sulfonic acid (0.24 M) or oxime-Osulfonate (0.1 M) at various pH values were conducted under the same conditions as with tyrosine. Reaction mixtures were analyzed using HPLC system 3. Ketoxime-O-sulfonates were prepared according to a published procedure (20). To an ice-cold methanol solution of the ketone, 10 mmol, and hydroxylamine-O-sulfonic acid, 10 mmol, was added 10 mmol of potassium acetate, saturated in aqueous methanol, while stirring. Stirring was continued for 10 more min; then the precipitate that formed was filtered and washed with ice-cold water followed by methanol and then dried. The oxime-O-sulfonates were not recrystallized from water since this could lead to their hydrolysis to oximes. Instead, the precipitated oxime-O-sulfonate was washed with copious amounts of cold methanol. As determined by NMR analysis, this step removes any oxime formed in the reaction as well as any unreacted hydroxylamine-O-sulfonate. The yields were 4070%, depending on the starting ketone. 1H NMR spectra of oxime-O-sulfonates were obtained using a Bruker 500 NMR spectrometer. 1H NMR spectra in DMSOd6: (1) acetoxime-O-sulfonate, δ 1.75 (2.5H, broad singlet, CH3), 1.84 (3H, broad singlet, CH3), 2.1 (1H, singlet, acetone formed perhaps by hydrolysis in DMSO), 3.4 (singlet, trace H2O present in DMSO-d6); (2) 2-heptanoxime-O-sulfonate, δ 0.85 (3H, triplet,
1422 Chem. Res. Toxicol., Vol. 10, No. 12, 1997
Figure 1. Hydrodynamic voltammetry of 3-aminotyrosine. A constant amount of 3-aminotyrosine (6.15 ng/injection) was chromatographed using a Whatman Partisil 5 SCX column (45 × 250 mm) eluted at a flow rate of 1 mL/min with 25 mM sodium phosphate, pH 3.4, containing 2% methanol. EC detector response was measured at various electrode potentials, and the peak areas were calculated and plotted against the applied potentials.
Sodum and Fiala
Figure 2. HPLC profile of some standard tyrosine derivatives. (A) Cation-exchange HPLC profile using HPLC system 2. The three standard compounds were injected simultaneously; UV and EC are the respective detector responses. (B) Ion-pair HPLC profile using HPLC system 1. In 1UV, only 3-nitrotyrosine was injected. This compound does not give a significant EC response at 475 mV. In 2UV and EC, tyrosine, 3-aminotyrosine, and 3-hydroxytyrosine were injected simultaneously. Under these conditions, tyrosine is not significantly electrochemically active. UV, UV detector response at 275 nm; EC, electrochemical detector response at 475 mV.
7-CH3), 1.29 (4H, multiplet, 6-CH2, 5-CH2), 1.45 (2H, quintet, 4-CH2), 1.75 (3H, singlet, 1-CH3), 2.15 (2H, triplet, 3-CH2), 3.4 (singlet, trace H2O present in DMSO-d6); (3) acetophenoximeO-sulfonate, δ 2.1 (1H, singlet, acetophenone formed perhaps by hydrolysis in DMSO), 2.2 (3H, singlet, CH3), 3.4 (singlet, trace H2O present in DMSO-d6), 7.4 (3H, multiplet, 3′-, 4′-, 5′-ArH), 7.7 (2H, multiplet, 2′-, 6′-ArH). Reaction of Tyrosine with Hydroxylamine-O-sulfonic Acid and Acetoxime-O-sulfonate. Hydroxylamine-O-sulfonic acid (20 mg, 0.18 mmol) or acetoxime-O-sulfonate (15.0 mg, 0.08 mmol) was added to a solution of L-tyrosine (1.0 mg, 0.005 mmol) in 1 mL of either 0.2 M acetate buffer at pH 4.7 or phosphate buffer at pH 6.4-7.0, and the mixture was shaken in an incubator at 37 °C for 4 h. Reaction mixtures were analyzed using HPLC systems 1 and 2.
Results Detection of 3-Aminotyrosine. Hydrodynamic voltammetry of 3-aminotyrosine, illustrated in Figure 1, shows that the compound is oxidized relatively easily at the glassy carbon electrode of the electrochemical detector. To analyze for 3-aminotyrosine in protein hydrolysates, we developed a cation-exchange HPLC system as well as an ion-pair method; the latter utilized n-heptanesulfonate as the counterion. With both systems, the electrochemical detector potential was set at 475 mV; at this potential neither 3-nitrotyrosine, a nitrosation and nitration product of tyrosine in vitro and in vivo (21-23), nor tyrosine gives a significant response. The latter is detected in protein hydrolysates by UV light absorption at 275 nm; at 475 mV the electrochemical detector is ∼104 times less sensitive to tyrosine than to 3-aminotyrosine. Figure 2A,B shows respectively the elution pattern of standard tyrosine and tyrosine derivatives by cationexchange HPLC and ion-pair HPLC. The more basic 3-aminotyrosine is retained more strongly on the cationexchange column than tyrosine or 3-nitrotyrosine (Figure 2A), whereas on the reverse-phase column (Figure 2B) the relatively less polar 3-nitrotyrosine is retained more strongly. With either HPLC mode, a detection level of ∼30 fmol of 3-aminotyrosine/injection is achievable with the electrochemical detector. 3-Hydroxytyrosine, also a product of the reaction of tyrosine with hydroxylamineO-sulfonate in vitro, elutes at ∼10 min in the ion-pair
Figure 3. Ion-pair HPLC profiles of tyrosine reaction products at pH 5.0 using HPLC system 2: trace 1EC, reaction carried out with acetoxime-O-sulfonate; trace 2EC, reaction carried out with hydroxylamine-O-sulfonate and diluted 50 times. UV, UV detector response at 275 nm; EC, electrochemical detector response. Table 1. Reactions of Hydroxylamine-O-sulfonate and Acetoxime-O-sulfonate with Tyrosine producta (nmol) reactant
pH 3-aminotyrosine 3-hydroxytyrosine
hydroxylamine-O-sulfonate 5.0 6.4 6.9 acetoxime-O-sulfonate 5.0 7.0
307.0 97.0 0.67 1.75 0.13
44.0 14.4 0.43 0.14 0.11
a Amount formed in 1.0 mL of mixture at 37 oC in 4-5 h. See Experimental Procedures for details.
system (Figure 2B) and is detectable due to its electrochemical activity under these conditions. In the cationexchange system 3-hydroxytyrosine elutes very early, near the void volume, and is obscured by noise (not shown). Amination of Tyrosine and Guanosine in Vitro. Acetoxime-O-sulfonate and hydroxylamine-O-sulfonate, intermediates in the proposed mechanism for the activation of 2-nitropropane, react with tyrosine at pH 5.0 and 6.9 to give 3-aminotyrosine (Figure 3 and Table 1). The reactivity of tyrosine with hydroxylamine-O-sulfonate at neutral pH is several times lower than that of guanosine (shown in Table 2), but at acidic pH both tyrosine and
Tyrosine Amination by Nitroalkanes and Acetoxime
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1423
Table 2. Reactions of Hydroxylamine-O-sulfonate and Oxime-O-sulfonates with Guanosine producta (nmol) reactant
pH
8-aminoguanosine
8-oxoguanosine
N1-aminoguanosine
hydroxylamine-O-sulfonate
5.0 6.9 8.0 5.0 7.0 8.0 5.0 7.0 5.0 7.0
356.5 33.6 8.6 1.1 0.04 0.11 0.11 nd 0.13 0.04
52.0 16.0 0.41 0.23 0.18 0.3 0.3 nd 0.8 1.22
not determined 80.8 97.6 ndc 2.32 7.73 nd nd nd nd
acetoxime-O-sulfonate acetophenoxime-O-sulfonate 2-heptanoxime-O-sulfonate
a Amount formed in 1.0 mL of mixture at 37 oC in 4-5 h. See Experimental Procedures for details. b Corrected for basal amount of 8-oxoguanosine in commercial preparation of guanosine. c Not detectable.
guanosine are almost equally reactive. Guanosine gives 10 times less 8-aminoguanosine at pH 7.0 than at pH 5.0. One reason for this lower reactivity at neutral pH could be a decreased production of NH2+; alternatively, it is possible that 3-aminotyrosine and 8-aminoguanosine react with the hydroxylamine-O-sulfonate to further products which were not detected by the present methods. In addition, as reported by other workers (13), at higher pH values hydroxylamine-O-sulfonate reacts preferentially with the N1 rather than the N7 or C8 of guanosine to give the N1-aminoguanosine adduct. N1Aminoguanosine is not electrochemically active under the conditions we employ. In an earlier report (11) we proposed a mechanism to explain the simultaneous formation of 8-oxoguanosine and 8-aminoguanosine in the reaction of hydroxylamine-O-sulfonate with guanosine. In the reactions of tyrosine with hydroxylamineO-sulfonate, we analogously observe the formation of 3-hydroxytyrosine along with 3-aminotyrosine. This is probably due to further reaction of 3-aminotyrosine with hydroxylamine-O-sulfonate. The reactions of acetoxime-O-sulfonate with tyrosine and guanosine to give aminated and hydroxylated tyrosine and guanosine derivatives were found to be pHdependent in the same way as reactions with hydroxylamine-O-sulfonate. Formation of more 8-aminoguanosine in reactions at acidic and basic pH than at neutral pH indicates that the hydrolysis of acetoxime-O-sulfonate to hydroxylamine-O-sulfonate is catalyzed by mild acid and base. The formation of N1-aminoguanosine in the reaction of guanosine with acetoxime-O-sulfonate at pH 8.0 also suggests strongly that the amination occurs through hydrolysis to hydroxylamine-O-sulfonate. O-Sulfonates of other ketoximes such as acetophenoxime and 2-heptanoxime, which too have the potential to form hydroxylamine-O-sulfonate, also react with guanosine to give 8-amino- and 8-oxoguanosines (Table 2) providing further evidence that other secondary nitroalkanes follow the same activation pathway as 2-nitropropane. Amination of Tyrosine in Vivo. To determine if, and to what extent, 2-nitropropane and related secondary nitroalkanes induce the amination of tyrosine in vivo, F344 male rats were treated with the chemicals once by ip injection, sacrificed after 6 h, and livers were removed for preparation of cytosol. Representative cation-exchange HPLC profiles of hydrolyzed liver cytosol preparations obtained from rats treated with vehicle control or 2-nitropropane are shown respectively in Figure 4A,B. Representative ion-pair HPLC profiles of hydrolyzed liver cytosol proteins from control rats or rats treated with 2-nitropropane are shown in Figure 5. As summarized in Table 3, only the secondary nitroalkanes, 2-nitropro-
Figure 4. Representative cation-exchange HPLC profiles of rat liver cytosol protein hydrolysates obtained from control rats treated with vehicle only (A) or with 2-nitropropane (B). Traces 2EC represent sample cochromatographed with 3-aminotyrosine standard. UV, UV response at 275 nm; EC, electrochemical detector response at 475 mV. Note the small EC peak in the control hydrolysate (A) at ∼15 min which coelutes with 3-aminotyrosine standard.
Figure 5. Representative ion-pair HPLC profiles of rat liver cytosol protein hydrolysates obtained from control rats treated with vehicle only (1EC) or with 2-nitropropane (2EC). UV, UV response at 275 nm (essentially identical for the two hydrolysates; only the trace obtained with control cytosol is shown); EC, electrochemical detector response at 475 mV.
pane, 2-nitrobutane, and 3-nitropentane, which had previously been shown to be hepatocarcinogens and substrates of rat liver aryl sulfotransferase (7), induced the amination of tyrosine in liver cytosol. The noncarcinogenic (7, 9) and/or nongenotoxic (7) primary nitroalkanes, 1-nitropropane, 1-nitrobutane, and 1-nitropentane, were inactive in this respect. Acetoxime, which qualitatively induces the same nucleic acid modifications as 2-nitro-
1424 Chem. Res. Toxicol., Vol. 10, No. 12, 1997 Table 3. Levels of 3-Aminotyrosine in Liver Cytosolic Proteins of Male F344 Rats Treated with Nitroalkanes or Acetoxime chemical administered
3-aminotyrosine/103 Tyra
vehicle vehicleb 1-nitropropane 1-nitrobutane 1-nitropentane acetoxime 2-nitrobutane 3-nitropentane 2-nitropropane 2-nitropropaneb 2-nitropropanec
e0.03 ( 0.03 e0.01 ( 0.01 e0.01 ( 0.002 e0.04 ( 0.003 e0.06 ( 0.01 0.53 ( 0.03 0.62 ( 0.03 0.11 ( 0.02 1.46 ( 0.07 1.23 ( 0.08 0.95 ( 0.13
a Means ( SD of at least three independent determinations each using one rat. Rats were killed 6 h after treatment except where noted. b Rats were sacrificed 18 h after treatment. c Rats were sacrificed 23 h after treatment.
propane (16) and is a liver tumorigen in rats (15), also produced 3-aminotyrosine, as expected. It should be noted, however, that the administered dose of this tumorigen was 3.3 times that of the nitroalkanes. Among all the nitroalkanes tested, 2-nitropropane induced the highest level of tyrosine amination in vivo; 1.5 mol of 3-aminotyrosine/103 mol of tyrosine was detectable in liver cytosol 6 h after treatment. This level decreased by ∼16% in 18 h and by ∼35% in 23 h. In the case of liver cytosols from animals treated with the primary nitroalkanes, or with aqueous Emulphor vehicle, a small peak due to an electrochemically active species was routinely observed at or very near the elution time of 3-aminotyrosine (Figures 4A and 5). In the case of cation-exchange HPLC, the peak was always observed and cochromatographed with 3-aminotyrosine standard. In the case of ion-pair HPLC (Figure 5, trace 1EC) the baseline obtained with the electrochemical detector was not as smooth as with cation-exchange HPLC, and the peak at or near the elution time of 3-aminotyrosine standard was not always well-defined. It is not possible at this time to definitely state whether this peak actually represents 3-aminotyrosine, but identity is not excluded. It would be of considerable interest if low, basal levels of 3-aminotyrosine were present in the proteins of the liver and other organs. One possible source for 3-aminotyrosine might be the reduction of 3-nitrotyrosine which is produced by endogenous nitrosating and nitrating agents such as nitrite, nitric oxide, and peroxynitrite (21-23). An alternative and perhaps even more intriguing possibility is the existence of an endogenous aminating species. These aspects are being pursued.
Discussion Detection of 3-aminotyrosine in the liver proteins of rats treated with 2-nitropropane or other secondary nitroalkanes represents strong support for the hypothesis that secondary nitroalkanes indeed undergo activation in vivo to a reactive aminating species, most probably the unsubstituted nitrenium ion, NH2+. The nonenzymatic reactions of acetoxime-O-sulfonate and hydroxylamine-O-sulfonate, proposed intermediates in the activation pathway of 2-nitropropane, with tyrosine and guanosine to give 3-aminotyrosine and 8-aminoguanosine, respectively (Tables 1 and 2), provide further positive evidence for the activation mechanism proposed in Scheme 1. The pH-dependent aminating property of acetoxime-O-sulfonate, which is strikingly similar to that
Sodum and Fiala
of hydroxylamine-O-sulfonate, indicates that amination proceeds through hydrolysis of acetoxime-O-sulfonate to hydroxylamine-O-sulfonate as proposed in Scheme 1. Clearly, other oxime-O-sulfonates would also be expected to act as aminating agents, irrespective of the alkyl groups attached to the carbon atom of the oxime function. The results from the in vitro reactions (Table 2) show that it is possible to aminate guanosine with various oxime-O-sulfonates. The yields are low, possibly because the hydrolysis of oxime-O-sulfonates to hydroxylamineO-sulfonic acid is slow and hydrolysis to oximes and ketones, which are not aminating agents, is probably a competing reaction. Oximes were isolated in quantitative yields by others during reactions of ketones with hydroxylamine-O-sulfonic acid when water was used to isolate the products (24). The formation of 3-hydroxytyrosine (L-DOPA) from tyrosine in vivo may be significant since 3-hydroxytyrosine is a metabolite of tyrosine and a precursor to catecholamine neurotransmitters. However, in the present work it was not possible to determine the nitroalkaneinduced hydroxylation of tyrosine in vivo because the conditions used during the hydrolysis of protein gave rise to high artifactual levels of 3-hydroxytyrosine, even in control liver samples. The biological significance of 3-aminotyrosine, which we have shown to be produced by the secondary nitroalkane-induced amination of protein tyrosine, is unknown. This modified amino acid has not, to our knowledge, been reported either as naturally occurring or as an in vivoinduced modification of tyrosine. Aminophenols are known to exert toxic effects such as depletion of thiols, methemoglobinemia, and hepatic and renal toxicity (25), although p-aminophenols are more toxic than o-aminophenols (26). Whether 3-aminotyrosine plays a significant role in the toxicity of secondary nitroalkanes and certain ketoximes, or if it has a cocarcinogenic role, remains to be investigated. Various important proteins, including DNA topoisomerases, tyrosine kinases, ribonucleotide reductase, purine nucleoside phosphorylase, and the product of the protooncogene c-met, have tyrosine at the active site, participating directly in the activity of the protein, or in close proximity to the active site, assisting the catalytic activity (27-31). Nitration of tyrosine has been used frequently as a tool for studying the role of tyrosine in enzymatic activity, but amination of tyrosines has not been employed extensively for this purpose. Nitration of tyrosine in vitro has been shown to inhibit the esterification of model peptides (32, 33), but the effects of amination of tyrosine are unknown. Compared to 3-nitrotyrosine, amination of tyrosine at the 3 position of the ring does not affect the pKa of the tyrosine phenolic group and exerts less steric hindrance. If 3-aminotyrosine does affect enzyme activity, it might be because of an additional amino functional group which protonates with a pKa of 4.5 (34) and has the potential for additional hydrogen-bonding and electrostatic interactions. The amination of tyrosine in ribonuclease A decreased enzymatic activity by 95%, in R-amylase by 46%, and in ovine placental lactogen by 70% (35-37). However, speculations regarding the possible biological effects of secondary nitroalkane-induced tyrosine amination must be tempered by the observation that this modification occurs at a frequency of less than 2 residues/1000 tyrosine residues in the liver cytosol of 2-nitropropane-treated rats (Table 3).
Tyrosine Amination by Nitroalkanes and Acetoxime
Modification of hemoglobin by carcinogens has been extensively investigated. For instance, hemoglobin adducts of environmental carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (a product of cigarette smoke), acrylonitrile, aromatic hydrocarbons such as styrene, and arylamines such as 4-aminobiphenyl have been developed as biomarkers for assessment of exposure (38-44). The hepatocarcinogen 2-(acetylamino)fluorene which also undergoes aryl sulfotransferasemediated activation to produce a reactive nitrenium ion has been shown to react with not only DNA but also methionine of liver protein (45). None of these carcinogens, however, has yet been shown to modify the tyrosine residues of this or other proteins. The biological significance of the secondary nitroalkane-induced tyrosine amination and its possible application as a biomarker of secondary nitroalkane or ketoxime exposure are currently being investigated in our laboratory.
Acknowledgment. We thank Ms. Helen Chan, a student intern from MIT, for analyzing some of the protein samples. We also thank American Health Foundation animal biologists Mr. Terrence Baxter and Ms. Erica Frew for their help. Thanks are given to Jim Lin for recording the NMR spectra. This work was supported by NIEHS Grant ES03257.
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1425
(14)
(15)
(16)
(17)
(18)
(19)
(20) (21)
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