In Vitro DNA Deamination by α-Nitrosaminoaldehydes Determined by

Joseph H. Banoub, Russell P. Newton, Eddy Esmans, David F. Ewing, and Grahame Mackenzie. Chemical Reviews 2005 105 (5), 1869-1916. Abstract | Full ...
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Chem. Res. Toxicol. 2000, 13, 72-81

In Vitro DNA Deamination by r-Nitrosaminoaldehydes Determined by GC/MS-SIM Quantitation Misun Park and Richard N. Loeppky* Department of Chemistry, University of Missouri, Columbia, Missouri 65211 Received July 9, 1999

The deamination of DNA bases by three R-nitrosaminoaldehydes, butylethanalnitrosamine, methylethanalnitrosamine, and N-nitroso-2-hydroxymorpholine (NHMOR), the direct metabolite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides, oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively. The order of relative reactivities of the bases was as listed above. Deamination of cytosine to uracil was detected by the reaction of 32P-labeled oligonucleotide ([5′-32P]CGAT) followed by enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf thymus DNA was performed by a gas chromatography/mass spectrometry-selected ion monitoring method. Both the extent and the rate of the deamination reactions which occur by transnitrosation from the R-nitrosaminoaldehyde to the base were determined for formation of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant at low substrate levels.

Introduction

Scheme 1 1

N-Nitrosodiethanolamine 1 (NDELA), a potent liver carcinogen in experimental animals, is one of the most prevalent and abundant of the environmentally occurring nitrosamines (1, 2) because it can be readily formed by the nitrosation of ubiquitous compounds such as di- or triethanolamine and their derivatives. It is a common trace contaminant in cosmetics, personal care items, tobacco products, metalworking fluids, and pesticides (3-5). The chemistry and mode of biochemical activation of this compound have been the subject of considerable investigation, yet while becoming clearer, an understanding of the initial events in its carcinogenic activation is lacking (6, 7). Nitrosamines are known to require metabolic activation to exert their carcinogenic effect (8). While the mode of biotransformation has not been elucidated for all nitrosamines, many of them are activated through the R-hydroxylation process (8-10). The biochemistry of enzymatic R-hydroxylation mediated by various subgroups of cytochrome P450 has been studied extensively and is reasonably well-understood (10). However, only recently has there been any evidence that the carcinogenic activation of NDELA and similar β-hydroxynitrosamines involves R-hydroxylation (6, 7). All prior experimentation has pointed to an activation step involving the oxidation of the 2-hydroxyethyl group to an aldehyde, a process known to be mediated by mammalian liver * Corresponding author. E-mail: [email protected]. 1 Abbreviations: NDELA, N-nitrosodiethanolamine; NHEE, Nnitrosohydroxyethylethanalnitrosamine; NHMOR, N-nitroso-2-hydroxymorpholine; NHEG, N-nitroso(2-hydroxyethyl)glycine; BuNE, butylethanalnitrosamine; MeNE, methylethanlanitrosamine; ADH, alcohol dehydrogenase; gG, N1-2-NH2-glyoxal-guanine adduct; SIM, selective ion monitoring; T4 PNK, T4 polynucleotide kinase; SVP, snake venom phosphodiesterase; DMP, Dess-Martin periodinane; TeC, 5′-CGAT DNA oligotetramer; TeA, 5′-ATCG DNA oligotetramer; TeG, 5′-GTAC DNA oligotetramer; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; dUMP, deoxyuridine phosphate; dIMP, deoxyinosine phosphate.

alcohol dehydrogenase (ADH) as shown in Scheme 1. The R-nitrosaminoaldehyde generated by the oxidation of NDELA, N-(2-hydroxyethyl)-N-nitrosoethanal 2 (NHEE), preferentially exists as a more stable hemiacetal N-nitroso-2-hydroxymorpholine 3 (NHMOR), and further oxidation leads to the formation of N-(2-hydroxyethyl)N-nitrosoglycine 4 (NHEG), the isolated urinary metabolite of NDELA (11, 12). It has been shown that β-hydroxynitrosamines are substrates for liver alcohol dehydrogenase in vitro, being reversibly converted to the corresponding aldehydes (7, 13, 14). The resulting R-nitrosaminoaldehydes are highly reactive compounds possessing the unique ability of transferring their nitroso groups to other amines, namely, transnitrosation (12-15), resulting in nitrosamine formation from secondary amines and diazonium ion formation and thence deamination of primary amines. This reaction occurs rapidly in organic solvents and is slower in aqueous buffer to some extent due to the equilibrium formation of gem-diol by hydration of the aldehyde group. In organic solvents, the transnitrosation reaction is often accompanied by the formation of imines of glyoxal. In aqueous media, both the imines and their hydrolysis products, primary amines and glyoxal, are found (13, 14), but it is not yet known if or how these processes are linked. As shown in Scheme 2 for adenine 6, transnitrosation from the R-nitrosaminoaldehyde to one of the primary amino groups in DNA bases is expected to result in deamination (15) through the formation of diazonium ion 8, its hydrolysis to 9, and the generation of a carbonyl 10 through tautomerism.

10.1021/tx990126d CCC: $19.00 © 2000 American Chemical Society Published on Web 12/28/1999

DNA Deamination by R-Nitrosaminoaldehydes Scheme 2

A number of the chemical and biological properties of NHMOR have been investigated after it was recognized as a probable important intermediate in the activation of NDELA (11, 12, 15). Chung and Hecht showed that deoxyguanosine reacts with NHMOR to form the cyclic 1,N2-glyoxal-deoxyguanosine 11 (gG) adduct (16).

As mentioned above, because NHMOR is a “masked” R-nitrosaminoaldehyde, it could deaminate the primary amino groups in DNA. The deamination process would change base-pairing characteristics or result in crosslinking, which could cause critical damage in DNA without leaving carbon fragments in the DNA backbone. The delineation of the role that NHMOR plays in NDELA activation requires that its ability to deaminate the NH2containing bases in DNA be defined since the level of radio-carbon incorporation from NDELA into DNA has been reported to be very low (17, 18). We have previously shown the deamination of guanosine by NHMOR and N-butylnitrosaminoethanal 5 (R ) nBu) (BuNE), as well as the formation of glyoxal-guanosine adducts 11 (14, 19, 20). In this paper, we demonstrate the deamination of DNA bases in oligonucleotides and calf thymus DNA by NHMOR and other R-nitrosaminoaldehydes. Quantitative analysis of deaminated bases performed by the GC/ MS-selective ion monitoring (SIM) method revealed that these nitrosamines deaminate guanine, adenine, and cytosine and that the deamination of guanine by NHMOR can be considered significant at the level of human environmental exposure to NDELA, its metabolic precursor.

Materials and Methods Chemicals and Instruments. Chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) or other commercial sources and were purified by conventional procedures, if necessary. DNA oligotetramers were prepared with an automatic DNA synthesizer by L. J. Forrest of the Department of Microbiology, University of Missouri. Calf thymus DNA, DNA mononucleotides, DNA bases, and other related biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). [1,3-15N2]Xanthine was kindly provided by J. Wishnok and S. R. Tannenbaum of the Department of Chemistry, Massachusetts Institute of Technology (Cambridge, MA). [γ-32P]ATP (3000 Ci/ mmol) was purchased from Dupont (Boston, MA). T4 polynucle-

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 73 otide kinase (T4 PNK) was obtained from United States Biochemicals (Cleveland, OH), and SVP (snake venom phosphodiesterase) was from Worthington Biochemical Co. (Freehold, NJ). 1H and 13C NMR spectra were recorded on either a Bruker AMX-500 (500 MHz for 1H and 125.8 MHz for 13C) or a Bruker ARX-250 (250 MHz for 1H and 62.9 MHz for 13C) spectrometer. HPLC was performed with a Waters instrument consisting of a model 510 solvent delivery system, a model 710B WISP autosampler, a model 490 multiwavelength UV detector, and HPLC control software of Maxima 820 or Millenium. An A-200 Flo-one/beta radioactive flow detector (Radiomatic Instruments & Chemical Co., Tampa, FL) was coupled to the HPLC system for radiochromatography. A programmable FC 203 Gilson fraction collector (Gilson Medical Electronics, Inc., Middleton, WI) was utilized for collecting eluted samples from the HPLC system. Gas chromatography/mass spectrometry (GC/MS) analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a Hewlett-Packard 5970 Series mass selective detector at an ionization voltage of 70 eV and controlled with Hewlett-Packard 59970 Chemstation software. Caution: Nitrosamines are potent chemical carcinogens and should be handled with extreme caution. A solution of anhydrous HBr in glacial acetic acid (10%) was used to decontaminate glassware and other washable items exposed to nitrosamines. Synthesis of r-Nitrosaminoaldehydes. Dess-Martin periodinane (DMP) was prepared as described in the literature (21). A dried 50 mL flask equipped with a magnetic stir bar, a septum, and a nitrogen balloon was charged with the starting nitrosamino alcohol (2.24 mmol, 1 equiv) (NDELA, methylethanolnitrosamine, or butylethanolnitrosamine) and dry CH2Cl2 (10 mL) to give a 0.22 M solution. DMP (1.05 g, 2.47 mmol, 1.1 equiv) dissolved in dry CH2Cl2 (10 mL) was added dropwise via a syringe. The reaction mixture was allowed to stir at room temperature for 1 h. The reaction was monitored by TLC. The white precipitate was removed by filtration, and the filtrate was diluted with diethyl ether (20 mL). A solution of Na2S2O3 (2.73 g, 17.29 mmol, 7 equiv of DMP) in 20 mL of saturated NaHCO3 was added, and the mixture was stirred for 10 min. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were concentrated on a rotary evaporator to give the desired product which was purified by flash chromatography. The spectral data of the resulting R-nitrosaminoaldehydes 5, N-nitroso-2-hydroxymorpholine (NHMOR, 3), N-methylnitrosaminoethanal (MeNE), or N-butylnitrosaminoethanal (BuNE), were in a good agreement with the literature (13). Reaction of DNA Oligotetramers with r-Nitrosaminoaldehydes. Stock solutions (2.0 M) of NHMOR, MeNE, and BuNE were prepared by dissolving the R-nitrosaminoaldehydes (26.4, 28.0, and 32.2 mg, respectively, 200 µmol each) in dimethylformamide (DMF, 100 µL). The 5′-GTAC tetramer (TeG, 990 µg, 0.79 µmol) was dissolved in sterilized water (100 µL) to give 7.9 nmol/µL of stock solution. The TeG solution (20 µL, 160 nmol) was mixed with the NHMOR or BuNE stock solution (6 µL, 12 µmol) in 100 µL of borate (0.15 M, pH 9.0) or phosphate (50 mM KH2PO4, pH 7.4) buffer. The mixture was incubated at 50 or 37 °C for 24 h and then hydrolyzed with diluted HCl (1.0 M, 100 µL) at 70 °C for 1 h. The hydrolysate was concentrated to dryness on a speedvac at room temperature. The residue was extracted with CHCl3 (4 × 200 µL) to remove excess nitrosamines and injected into the HPLC system with a µBondapak C-18 column (10 µm, 30 cm × 3.9 mm, Waters) using a gradient program (I) as follows: solvent A, 0.1 M NaH2PO4 (pH 5.5); solvent B, methanol; 100% A from 0 to 5 min, gradient to 8% B over the course of 20 min and then to 30% B over the course of 5 min; flow rate, 1.2 mL/min. Preparation of 32P-Labeled Oligonucleotides. To a 1.5 mL microcentrifuge tube containing 10× phosphorylation buffer [0.5 M Tris-HCl (pH 8.0)/0.1 M MgCl2 and 0.015 M spermidine, 20 µL] was added the DNA oligotetramer solution (20 pmol/µL, 2 µL, 40 pmol), 5′-CGAT (TeC), 5′-ATCG (TeA), or 5′-GTAC

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(TeG), followed by 3000 Ci/mmol [γ-32P]ATP (6 µL, 60 µCi, 20 pmol). T4 PNK (30 units/µL) was diluted to a concentration of 3 units/µL with Tris-HCl buffer (50 mM, pH 8.0) immediately prior to use. The diluted T4 PNK solution (6 µL, 18 units) was added to the reaction tube, and the final volume was adjusted to 100 µL with autoclaved water. The mixture was incubated at 37 °C for 30 min. The labeling reaction was terminated by adding EDTA solution (0.5 M, 1 µL) followed by heating at 65 °C for 5 min. The labeled tetramer was purified by HPLC employing a Radiomatic detector. A Zorbax ODS column (4.6 mm × 25 cm) was used, and the following gradient program (II) was employed: solvent A, 50 mM KH2PO4 (pH 5.2); solvent B, methanol; gradient from 15 to 20% B over the course of 15 min, then to 30% B over the course of 10 min, and 30% B for 2 min; flow rate, 1.4 mL/min. The 32P-labeled tetramer peak was collected on a fraction collector and then loaded on a C-18 SepPak cartridge (Waters) for desalting. The cartridge was washed with distilled water (10 mL) to remove buffer salts, and the labeled tetramer was eluted with 75% acetonitrile in water (4 mL). The resulting tetramer solution was divided into six microcentrifuge tubes and concentrated to dryness on a speedvac to give about 2 pmol of 32P-labeled tetramer in each tube. Reaction of the 32P-Labeled Tetramer with HNO2. The 32P-labeled tetramer (about 2 pmol) in a 1.5 mL microcentrifuge tube was mixed with NaNO2 solution (5.0 M, 20 µL) and KH2PO4 buffer (50 mM, pH 3.0, 200 µL). The reaction mixture was shaken at 50 °C for 24 h, and then concentrated to dryness on a speedvac. The residue was dissolved in SVP buffer (100 µL). A SVP stock solution (1.52 units/µL) was prepared by dissolving the enzyme (152 units) in SVP buffer [Tris-HCl buffer (50 mM, pH 8.8) containing 3 mM MgCl2 (90 µL)] and BSA solution (bovine serum albumin, 10 µg/µL, 10 µL). The stock solution was stored at -28 °C. Total enzymatic hydrolysis was performed by adding SVP stock solution (2 µL, 3 units) followed by shaking at room temperature for 20 h. Partial hydrolysis was carried out for comparison with the following procedure. The 32P-labeled tetramer (about 2 pmol) was dissolved in SVP buffer (100 µL) and mixed with SVP stock solution (0.4 µL) for 5 s at room temperature. The reaction was stopped by adding 0.1 M EDTA (3 µL) followed by heating at 65 °C for 1 min. The hydrolysates were analyzed by HPLC with a Zorbax ODS column employing the following gradient program (III): solvent A, 50 mM KH2PO4 (pH 5.2); solvent B, acetonitrile; gradient from 2 to 30% B over the course of 30 min and 30% B for 10 min; flow rate, 1.2 mL/min. Reaction of the 32P-Labeled Tetramer with r-Nitrosaminoaldehydes. The 32P-labeled tetramer in a 1.5 mL microcentrifuge tube was mixed with R-nitrosaminoaldehyde stock solution (2.0 M, 5 µL) and borate buffer (0.15 M, pH 9.0, 100 µL). The reaction mixture was shaken at 50 °C for 24 h and then extracted with CHCl3 (4 × 200 µL) to remove unreacted nitrosamines. The mixture was concentrated on a speedvac to dryness, and the resulting residue was dissolved in SVP buffer (100 µL) and hydrolyzed with SVP (2 µL, 3 units) at room temperature for 20 h. Control experiments were performed without nitrosamine. The hydrolysate was analyzed by HPLC using gradient program III. Deamination in Calf Thymus DNA Determined by GC/ MS-SIM (22). Although the analytical methodology used for the accurate measurement of DNA deamination, except for note variations, is essentially the same as that published by Nguyen et al., the proper analytical conditions were carefully worked out in our laboratory by the optimization of each step with respect to precision, reproducibility, and accuracy through the use of standards. The standard solutions of DNA bases and modified bases were prepared by dissolving adenine, guanine, hypoxanthine, and xanthine (2.8, 3.1, 2.8, and 3.1 mg, respectively, 20 µmol) in DMSO (600 µL). The standard solution of [15N2]xanthine was prepared by dissolving the sample (1.4 mg, 9.1 µmol) in DMSO (600 µL) to give a concentration of 15.15 nmol/µL. The standard solution (10 µL) was added into a conical glass vial and dried on a speedvac at 50 °C. The residue was

Park and Loeppky dissolved in dry acetonitrile (100 µL), and then dry pyridine (20 µL) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, 80 µL, 0.37 mmol) were added. The reaction vial was tightly sealed and heated at 100 °C for 1 h. The sample (1 µL) was analyzed by GC/MS under the following conditions. A fusedsilica capillary SPB-1 column, 30 m × 0.25 mm (Supelco), was used. The injector and transfer lines were kept at 280 °C. The temperature program was as follows; the initial temperature was 150 °C with an initial time of 2 min, and the temperature was increased to 220 °C at a rate of 10 °C/min, and then to 280 °C at a rate of 25 °C/min with a final time of 5 min. The samples were injected into the GC/MS apparatus with a low split rate. The base peaks of M-15 of the derivatized bases were observed: m/z 264 for di-TMS-adenine, m/z 352 for tri-TMS-guanine, m/z 265 for di-TMS-hypoxanthine, m/z 353 for tri-TMS-xanthine, and m/z 355 for tri-TMS-[15N2]xanthine. A mixture of standards was derivatized, and only the base peaks were monitored in the SIM mode which provides a more accurate measurement of the abundance of ions of interest. Dwell times were 20 ms per ion. The retention times on the GC/MS apparatus were 8.2 min for hypoxanthine, 8.9 min for adenine, 10.3 min for xanthine and [15N2]xanthine, and 11.1 min for guanine. Calf thymus DNA (2.5 mg) was dissolved in KH2PO4 buffer (50 mM, pH 7.4, 0.5 mL) and the solution mixed with R-nitrosaminoaldehydes (20 mg in 10 µL of DMF). A control DNA sample was prepared with buffer solution and DMF. The mixture was shaken at 50 °C for 48 h. A part of the reaction mixture (100 µL) was taken and dried on a speedvac. This step differs for the method of Nguyen et al. (22), who utilized ethanol to precipitate the DNA. Control experiments consistently showed that smaller amounts of xanthine were obtained when ethanol precipitation was used compared to the direct speedvac drying of the sample. The residue was extracted with CHCl3 (4 × 200 µL) and then dissolved in diluted HCl (1.0 M, 100 µL). The mixture was hydrolyzed at 70 °C for 1 h, and the hydrolysate was directly injected into the HPLC system with a Synchropak RP-P-100 column, 4.6 mm × 25 cm (Alltech), using the following gradient program (IV): solvent A, 0.1 M ammonium acetate (pH 5.2); solvent B, methanol; 100% A from 0 to 15 min, gradient to 30% B over the course of 5 min, and 30% B for 5 min; flow rate, 1.0 mL/min. The eluted fractions corresponding to adenine, guanine, hypoxanthine, and xanthine were collected on a fraction collector. The combined fractions were neutralized to pH 7 with ammonium hydroxide and concentrated to a volume of 150-200 µL on a cold trap (dry ice/acetone) rotary evaporator in a vacuum. The resulting liquid was transferred to a conical glass vial and dried on a speedvac at 50 °C. The residue was dissolved in dry acetonitrile (100 µL) and dry pyridine (20 µL), and then derivatized with MSTFA (80 µL, 0.37 mmol) by heating at 100 °C for 1 h. The derivatized sample (1 µL) was injected into the GC/MS system under the same conditions as described above. Base peaks at M-15, m/z 264 for di-TMS-adenine, m/z 265 for di-TMS-hypoxanthine, m/z 352 for tri-TMS-guanine, and m/z 353 for tri-TMS-xanthine, were monitored. Dwell times were 20 ms per ion. Quantitation of Deamination by GC/MS-SIM (22). DNA samples and controls were prepared as described above with an R-nitrosaminoaldehyde concentration ratio of 64 µmol/mg of DNA. After acidic hydrolysis, the hydrolysate was combined with a [15N2]xanthine standard solution (2 µL, 30.3 nmol) and injected onto the HPLC system (program IV). The modified and unmodified base fractions were collected, neutralized, and dried for derivatization. The dried sample was dissolved in dry acetonitrile (60 µL) and dry pyridine (40 µL), and then derivatized with MSTFA (100 µL, 0.46 mmol) at 100 °C for 1 h. The derivatized samples were analyzed by the GC/MS-selected ion monitoring (SIM) process. Base peaks at M-15 were monitored with a dwell time of 20 ms per ion. The quantities of modified and unmodified bases were calculated by comparing the abundances of M-15 ions of the derivatized bases with that of the internal standard (m/z 355 for tri-TMS-[15N2]xanthine). The method was validated by using a standard curve and by

DNA Deamination by R-Nitrosaminoaldehydes determining the ratio of bases (unmodified) in the DNA control sample in comparison with that reported in the literature (23), and excellent agreement was observed. A calf thymus DNA solution (5.0 mg/mL) was prepared in KH2PO4 buffer (50 mM, pH 7.4) and sonicated for 2 min to break the DNA into small pieces. Stock solutions (16.0 M) of R-nitrosaminoaldehydes were prepared by dissolving NHMOR, BuNE, and MeNE (0.21, 0.23, and 0.16 g, respectively, 1.6 mmol) in DMF (100 µL). (1) Time Dependence. The nitrosamine stock solution (2 µL, 32 µmol) was mixed with a DNA solution (100 µL, 0.5 mg) and shaken at 37 °C for the desired length of time (1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h). Control experiments were performed via 4, 8, 12, 24, and 48 h incubations of DNA solutions without nitrosamine. (2) Concentration Dependence. Various amounts of the respective nitrosamine stock solutions (2, 4, 6, and 8 µL) were mixed with a DNA solution (500 µL) and incubated for 24 h at 37 °C. (3) Guanine Deamination by NHMOR at Lower Concentrations. Reaction mixtures were prepared with same procedure described above except for the use of a diluted NHMOR solution (1.0 or 0.1 M) to make the concentration ratios of nitrosamine 0.1, 0.2, 0.4, 0.6, 1.0, and 2.0 µmol/mg DNA, respectively. The mixtures were incubated for 4 h at 37 °C. The reaction mixtures were dried on a speedvac and then extracted with CHCl3 (5 × 200 µL). The residues were hydrolyzed at 70 °C with dilute HCl (2.0 M, 100 µL) for 1 h. The hydrolysates were combined with the [15N2]xanthine standard solution (2 µL, 30.3 nmol) and injected into the the HPLC system using elution program IV. The fractions containing xanthine and hypoxanthine were collected and derivatized according to the procedure described above. The derivatized samples were analyzed with the GC/MS-SIM method.

Results and Discussion Synthesis of r-Nitrosaminoaldehydes by DessMartin Oxidation. R-Nitrosaminoaldehydes have been commonly synthesized from the corresponding amine and R-chloroaldehyde acetal to give aminoacetal followed by nitrosation with isopropyl nitrite (13). The yield of the aminoacetal in the first step varies from 42 to 58%. In this synthetic procedure, isopropyl nitrite was used to nitrosate the aminoacetals, but the yields of the nitrosation range from 65 to 88%, which often requires a long reaction time, more than 48 h, to obtain a reasonable amount of the product. The resulting nitrosaminoacetals were treated with 6 M HCl to generate the corresponding nitrosaminoaldehydes which were extracted after neutralization with a saturated NaHCO3 solution. The yields of this final step were about 75% for relatively nonpolar aldehydes possessing bulky alkyl groups, but were only about 30% for highly water-soluble compounds such as N-methylnitrosaminoethanal 5 (R ) CH3) (MeNE) and NHMOR. In an attempt to overcome difficulties such as the low overall yield and time-consuming procedure, a direct oxidation of nitrosamino alcohols to their corresponding aldehydes was taken into consideration. The oxidation of alcohols to aldehydes or ketones has been carried out with a variety of methods, and one of the most effective and convenient methods is the Dess-Martin reaction which uses an organo-iodine compound (21, 24). The synthesis of the oxidizing agent, Dess-Martin periodinane (DMP), was carried out according to the known procedure (21) with 87% overall yield from the starting iodobenzoic acid. Application of this effective yet mild oxidation method to the preparation of R-nitrosaminoaldehydes reduces the synthesis procedure to two steps (Scheme 3). The starting nitrosamino alcohols 13, NDE-

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 75 Scheme 3

LA (R ) CH2CH2OH), methylethanol nitrosamine (R ) CH3), and butylethanolnitrosamine (R ) nBu), were prepared by the nitrosation of the corresponding amines 12 with sodium nitrite and glacial acetic acid in high yield. The oxidation of nitrosamino alcohols by DMP was carried out at room temperature in 0.02 M acetic acid. The reaction was complete within 1 h, providing quantitative conversion in all cases as monitored by TLC. In the case of NDELA, the formation of dialdehyde as a side product is considered to be prevented under acidic conditions by facilitating the ring closure of the resulting monoaldehyde to its hemiacetal, NHMOR 3. A neutral workup procedure using Na2S2O3 was employed to prevent the decomposition of base-sensitive R-nitrosaminoaldehydes. The yields of the R-nitrosaminoaldehydes after chromatographic purification are given in Scheme 3. The application of Dess-Martin oxidation to the synthesis of R-nitrosaminoaldehydes provides great advantages. The procedure is simplified, involving only nitrosation of amino alcohols and oxidation of the resulting nitrosamino alcohols to aldehydes. The overall yields are about 85% from the starting amine, which is a great improvement especially for highly water-soluble products, NHMOR and MeNE 5 (R ) CH3). These compounds have been of great interest in the study of nitrosamine carcinogenesis, and a large number of investigations are still underway. With this new method, the synthesis of R-nitrosaminoaldehydes has become more convenient and efficient. Chemistry of Deamination by r-Nitrosaminoaldehydes. The prior research and model studies on the properties of R-nitrosaminoaldehydes, and, in particular, their reactions with guanosine and deoxyguanosine, clearly lead to the expectation that these reactive Nnitroso compounds should react with DNA. The two expected transformations are deamination and glyoxalguanine (gG) adduct formation, but the reactions of NHMOR and other R-nitrosaminoaldehydes with DNA have not been reported. The focus of this work, therefore, was to determine whether these reactive aldehydes, NHMOR in particular, can deaminate DNA bases through transnitrosation. In a preliminary report or our work (19), we presented evidence which showed that R-nitrosaminoaldehydes deaminate DNA mononucleotides, dGMP, dAMP, and dCMP. The mononucleotides were reacted with either NHMOR 3 or BuNE (5, R ) nBu) in buffer (pH 7.4 or 9) and a small amount of DMF to facilitate dissolution of the mixture. After reaction for 48 h at 50 °C, the bases were released by heating in acid and subjected to HPLC. Appropriate controls were performed to test for deamination under conditions of hydrolysis. Both NHMOR and BuNE deaminated dGMP 14 and dAMP 6 to give xanthine 10 and hypoxanthine 16, respectively. The formation of glyoxal-guanine (gG) adduct 11 from dGMP was also observed in high yield (see Scheme 4). In all cases, BuNE produced more deamination than NHMOR. How-

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Chem. Res. Toxicol., Vol. 13, No. 2, 2000 Scheme 4

ever, the deamination of dCMP 15 to produce uracil 17 was not well-characterized by this method, which was due to the difficulty in hydrolyzing the glycosidic bond of pyrimidine nucleotides. The detection of dUMP, the deamination product of dCMP, from the reaction mixture was not successful by UV monitoring. It is known that the reactivity of cytidine toward deamination by nitrosating agents is much lower than that of guanosine and adenosine (25). To detect the deamination of dCMP, a more sensitive method was required. Reaction of DNA Oligotetramers with r-Nitrosaminoaldehydes. The use of individual mononucleotides clearly demonstrated that R-nitrosaminoaldehydes deaminate DNA bases and form the glyoxal-guanine adduct (19). These reactions were conducted under the conditions adjusted to provide the maximum rate of transnitrosation, which were different from physiological conditions. Since our interest lies in the carcinogenesis of the reactive aldehydes in biological systems, it is important to know whether the chemical transformations observed in the previous experiments would take place under conditions mimicking more natural biological conditions. To test this, R-nitrosaminoaldehydes were reacted with 5′-GTAC (TeG) under both physiological conditions (pH 7.4, 37 °C) and the reaction conditions used previously (pH 9, 50 °C) for comparison. The results showed no major difference in these two reaction conditions. The chromatogram of the reaction of NHMOR with TeG shows that xanthine and the glyoxal-guanine adduct are formed (data not shown). Similar observations were made for BuNE which appeared to produce more of the glyoxal-guanine adduct than xanthine. The deamination of adenine could not be observed under the chromatographic conditions used, since hypoxanthine overlaps with guanine. Attempts to separate the two bases within an appropriate pH range (4.5-7.5) for this analysis were not successful. Since the amount of TeG used and the yield of the products were very low, the HPLC/UV chromatograms were unable to provide clear results. Therefore, 32P-labeled DNA oligotetramers were used for more sensitive detection. Originally (26), we attempted to use oligotetramers to detect reactions at each kind of base by employing 5′-32P-labeled oligotetramers, containing one base of each type, followed by partial enzymatic hydrolysis after the separation of the modified oligomers. The initial separations are difficult, however, when reactive substrates produce a myriad of modifications (adducts). Moreover, adduction can impede

Park and Loeppky

enzymatic hydrolysis. Many of these problems can be overcome by the use of 5′-32P-labeled oligotetramers which employ a different base at the 5′-end of each oligomer, and total enzymatic hydrolysis of the 3′phosphodiester bonds is performed (26). Use of 5′-32P-Labeled DNA Oligotetramers. The tetramers 5′-CGAT (TeC), 5′-ATCG (TeA), and 5′-GTAC (TeG) were labeled with [γ-32P]ATP according to the standard procedure and purified by HPLC. These labeled tetramers were reacted with R-nitrosaminoaldehydes, NHMOR, BuNE, and MeNE, in borate buffer (pH 9) for 24 h at 50 °C or with HNO2 at pH 3.5 for 2 h (NO2- does not react with DNA under neutral or basic conditions). Total enzymatic hydrolysis followed by HPLC coupled using a radioflow detector permits the detection of modifications on the 5′-base. The chromatograms of the nucleotides from the reactions were compared with standards, and those from reactions with nitrous acid as well as the hydrolysate of the unreacted tetramer (control). In each case (data not shown), a partial enzymatic hydrolysis was performed as a control to ensure that peaks seen in the total hydrolyses were not due to oligomers of various sizes. The radiochromatograms for the reactions of TeC are shown in Figure 1. All radiochromatograms of the reactions of TeC with the R-nitrosaminoaldehydes showed the presence of dUMP, the deamination product of cytosine (after hydrolysis). It was observed in high yield in the reaction with nitrous acid. The deamination was not observed in the control experiment. Among the R-nitrosaminoaldehydes, BuNE exhibited higher reactivity than NHMOR and MeNE (data not shown). From the reactions of TeA with R-nitrosaminoaldehydes, deoxyinosine 5′monophosphate (dIMP), the deamination product of adenine, was also observed (data not shown). The intensity of dIMP peaks was very low, but clearly, no dIMP was detected in the control experiment. The reaction with nitrous acid exhibited a much higher yield of dIMP. The reactions of TeG with R-nitrosaminoaldehydes did not show any common adducts or modifications in the radiochromatogram compared to those observed in the nitrous acid reaction of TeG. For each R-nitrosaminoaldehyde, the radiochromatograms exhibited a large peak due to the gG adduct (comparison with standard) (20) which eluted before dGMP. The same peak was also observed in high yield from the reaction of glyoxal with TeG followed by enzymatic hydrolysis. The failure to observe [5′-32P]deoxyxanthosine monophosphate, the deamination product of G in TeG, in this case is attributed to either cleavage of the nucleosidic linkage under the workup and enzymatic hydrolysis conditions or, more probably, its being obscured by the gG adduct peak. With the use of 32P-labeled DNA oligotetramers, it was possible to detect the deamination of cytosine and adenine which were not well-characterized by UV detection. Quantitation of the Deamination in Calf Thymus DNA by GC/MS-SIM Method. Studies on the reactions of R-nitrosaminoaldehydes with DNA have shown that these reactive aldehydes are capable of deaminating DNA bases. The possible biological significance of these transformations obviously depends on the relative rate of DNA reactions compared to those of other processes, particularly those which act to detoxify the R-nitrosaminoaldehydes by oxidation to unreactive, readily excreted carboxylic acids. To elucidate biological consequences of deamination in relation with mutagenicity of the R-nit-

DNA Deamination by R-Nitrosaminoaldehydes

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 77

Table 1. Yields and Rates of Purine Deamination by Nitrosaminoaldehydes xanthinea aldehyde

nmol/mgc

NHMOR BuNE MeNE control

64.5 ( 0.8 121.2 ( 3 49.1 ( 3 1.4 ( 0.09

%

hypoxanthineb e

Vo

nmol/mgc

% deaminatedd

Voe

181 ( 23 125 ( 2 73 ( 4 -

7.9 ( 0.3 22.9 ( 0.4 5.6 ( 0.5 0.10 ( 0.02

0.9 ( 0.03 2.6 ( 0.05 0.10 ( 0.01 0.012 ( 0.003

5 ( 0.6 30 ( 0.7 7 ( 0.2 -

deaminatedd 10.4 ( 0.1 19.6 ( 0.5 5.6 ( 0.4 0.23 ( 0.01

a From guanine. b From adenine. c Yield in nanomoles per milligram of DNA at 48 h, pH 7.4, and 37 °C. d The percentage of deamination was estimated using the amounts of adenine and guanine in calf thymus DNA reported in the literature (23), which are 891.3 and 620.0 nmol/mg of DNA, respectively. e Picomoles of product per milligram of DNA per minute.

Figure 1. Radiochromatograms from the reactions of [5′-32P]CGAT (TeC) with the indicated compound, after enzymatic hydrolysis. (A) NHMOR; peaks at retention times > 19 min are partial hydrolysis products. (B) BuNE. (C) HNO2. (D) Control; 5′-32P-CGAT after total hydrolysis.

rosaminoaldehydes, a detailed quantitative investigation of R-nitrosaminoaldehyde-induced DNA deamination reactions was necessary. The quantitation of xanthine and hypoxanthine produced by the reaction of nitric oxide with DNA, RNA, or intact human cells has been reported by Nguyen and co-workers (22). The analysis was carried out using the GC/MS-SIM method after derivatizing the dried residue of xanthine and hypoxanthine collected from HPLC. Quantitation was carried out by comparing the abundances of the M-15 ions of the TMS-derivatized bases with that of the internal standard, [1,3-15N2]xanthine. This same analytical method was utilized by us for the quantitative determination of DNA deamination by R-nitrosaminoaldehydes.

To test the method and determine whether we could observe sufficient R-nitrosaminoaldehyde-mediated deamination of DNA under our experimental conditions, calf thymus DNA was reacted with NHMOR, BuNE, or MeNE (64 µmol/mg of DNA) under physiological conditions (pH 7.4, 37 °C) for 48 h. The reaction mixtures were prepared for HPLC after extracting the remaining nitrosamines with chloroform. The fractions containing adenine, guanine, hypoxanthine, and xanthine were collected and dried for derivatization. Adenine and guanine were collected from the control DNA sample to test the reliability of the method. The derivatized samples were analyzed by the GC/MS-SIM method. The relative abundancies of the base peaks of the deaminated bases compared to those of unmodified bases were calculated, which provided the following initial results: hypoxanthine/adenine, 0.2 for NHMOR, 0.1 for BuNE, and 0.1 for MeNE; xanthine/guanine, 0.5 for NHMOR, 0.7 for BuNE, and 0.3 for MeNE. The relative yields of deaminated bases from this experiment are in a good accordance with those we obtained from the deamination of mononucleotides. The ratio of adenine and guanine in control calf thymus DNA was observed to be 1:0.7, which agrees with the published data, 1:0.69 (23). Encouraged by these results, we carefully optimized our procedures to accurately determine the extent of deamination as a function of nitrosamine concentration and time. Each run involved the utilization of three samples at each time point or concentration. In every case, a determination of the purine base ratios in a standard DNA control sample was made, and we measured the individual concentrations of the hypoxanthine and xanthine in the control sample. As might be expected, no hypoxanthine was observed in the control samples at long run times, although low levels of xanthine were observed. This “natural” guanine deamination was very small compared to that produced by the nitrosamine at the same time point. By this means, the percent error as measured by the standard deviation of the means for all runs was in the range of 0.9-1%. Specific values are given in Table 1 where we report the initial rates of deamination and the yield of deamination product after incubation for 25 h at 37 °C and pH 7.4. The results provided by this GC/MS-SIM analysis clearly show that the deamination of bases in DNA, including that of adenine to form hypoxanthine which was not clearly visible in double-stranded DNA by the HPLC/UV method, by R-nitrosaminoaldehydes certainly occurs. The highest yield of deamination was observed in the reaction with BuNE. NHMOR was moderately reactive among the three compounds, and MeNE was the least reactive. A significant amount of deamination occurred in the reaction with NHMOR, a process which could occur in vivo. To obtain a better understanding of the biological importance of the deamination reaction induced by R-nit-

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Figure 2. Effect of nitrosamine concentration upon the quantity of xanthine produced from DNA within 24 h under physiological conditions. The plots are for the reciprocals of the variables as indicated by the axes labels at the left and bottom. The axes labels at the top and right give the corresponding concentrations of substrate and xanthine, respectively.

Scheme 5

rosaminoaldehydes, the extent of deamination as a function of nitrosamine concentration and time was determined. Concentration Dependence of Deamination. The dependence of the deamination on the R-nitrosaminoaldehyde concentration was studied by varying the amount of the aldehyde from 12.8 to 64.0 µmol/mg of DNA in 100 µL. The reaction mixtures were incubated for 24 h under physiological conditions. The results are shown in Figure 2 for the production of xanthine from the deamination of guanine. Similar data were observed for the deamination of adenine to give hypoxanthine, although the formation of hypoxanthine was 6-10 times slower than that of xanthine. While the yield of the deaminated base at a fixed time point increases with the concentration of the R-nitrosaminoaldehyde, the increase is not linear over the concentration range. Rather, a saturation phenomenon is observed where an increase in the concentration of the R-nitrosaminoaldehyde does not produce a proportional increase in the concentration of the deaminated base. This phenomenon was very reproducible and observed over several concentration ranges. These observations suggested that the yield of deaminated base at any time point could be represented by a function such as that shown for xanthine in eq 1 of Scheme 5, where a, b, and c are constants. Equations of this type are, for example, typical for adsorption processes where substrates are reversibly competing for sites, such as those in DNA where a transformation could occur. The recipro-

Park and Loeppky

Figure 3. Xanthine formation from the deamination of guanine in calf thymus DNA by NHMOR at low concentrations after 4 h at 37 °C. The inset shows the regression line for the doublereciprocal plot.

cal of eq 1 gives eq 2, which predicts that a plot of the reciprocal of the xanthine yield, at any time, versus the reciprocal of the initial aldehyde concentration should be linear. In fact, we found double-reciprocal plots of the yields of deaminated bases are linear as is shown in Figures 2 and 3. Because we do not know the mechanism of the deamination transformation at this time, we cannot know with certainty why the double-reciprocal plots are linear. There are, however, several reasonable explanations. For example, if we assume that the formation of a deaminated base is represented simply by eqs 3 and 4 of Scheme 5, and if it is assumed that changes in the R-nitrosaminoaldehyde and DNA are negligible with respect to the relatively low yield of the deaminated base (e.g., xanthine), conditions which certainly hold in our work, then it can be shown that eq 1 (and eq 2) holds where a ) k1k2t, b ) (k-1 + k2), and c ) k1. However, equations of this type can also be derived when the substrate enters into another process competitively which does not produce a deaminated base. This could involve either the formation of the gG adduct in a transformation which is independent of the deamination reaction or another reaction of the nitrosaminoaldehyde with the DNA, e.g., formation of Schiff base adducts or another undetermined transformation. While we have previously shown that glyoxal equivalents are generated in the transnitrosation reactions of R-nitrosaminoaldehydes (13-15, 19, 20), we have not shown that the transformations are actually coupled, although current research in our laboratory is directed at this point. To further establish the possible biological relevance of the deamination reaction, we have examined the deamination of guanine bases in DNA by NHMOR at concentrations lower than those utilized above. Prior work in other laboratories led us to conclude that adenine and cytosine deamination levels would be very low at real exposure levels due to the low reactivity (22, 27). Since guanine is more reactive than the other bases, and NHMOR is a known metabolite of NDELA, a carcinogen to which humans are exposed, the deamination of guanine was studied with lower NHMOR concentrations. Concentration-related increases in the level of xanthine formation after incubation for 4 h with varying concentrations of NHMOR, from 0.1 to 2.0 µmol/mg of DNA, measured by the GC/MS-SIM method are illustrated in Figure 3. Here again, a double-reciprocal plot was linear and the curve in Figure 3 was generated by transforma-

DNA Deamination by R-Nitrosaminoaldehydes

Figure 4. Time course of guanine deamination in calf thymus DNA by the three R-nitrosamino aldehydes. The inset shows the data for NHMOR-mediated deamination in the form of a plot of ln(1 - X/X∞) vs t (hours).

tion of the linear regression of the data in that form (R2 ) 0.98). A xanthine yield of 1.8 nmol/mg of DNA was formed from 0.1 µmol of NHMOR. The deaminated bases in human cells treated with nitric oxide in vitro have been detected by GC/MS-SIM quantitation at the level of 10-1 nmol/mg of DNA (22). Extrapolation of our data suggests that the same level of guanine deamination produced by NO could be achieved by a concentration ratio of approximately 10-2 µmol of NHMOR/mg of DNA. These results suggest that detection of DNA deamination by NHMOR in biological samples where the level of environmental exposure to its metabolic precursor, NDELA, has been high may be possible by the GC/MS-SIM method. However, as mentioned earlier, a significant amount of xanthine depurinates from the DNA backbone and this unknown quantity is excluded when DNA samples are prepared by the precipitation method. Because of this fact, accurate quantitative determination of xanthine in vivo could only be achieved after establishing appropriate procedures for DNA separation from biological samples. Time Dependence of Deamination. The effect of incubation time on the deamination reaction was examined by GC/MS-SIM quantitation. A calf thymus DNA solution (5.0 mg/mL) was prepared in phosphate buffer and sonicated to break the DNA into small pieces to make the solution homogeneous. The DNA solution was reacted with NHMOR, BuNE, or MeNE under physiological conditions. The concentration of the R-nitrosaminoaldehydes was 64.0 µmol/mg of DNA. The samples taken after incubation for 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h were analyzed for the amount of xanthine and hypoxanthine. Control samples were prepared by incubation of DNA solution without nitrosamine for 4, 8, 12, 24, and 48 h. Figure 4 shows the transformation time course for xanthine formation as a result of guanine deamination by the nitrosaminoaldehydes. It is evident from the inspection of Figure 4 that the reaction exhibits a saturation phenomenon. The greatest yield was obtained from BuNE. Plots of ln(1 - X/X∞) versus t, where X is the xanthine concentration at time t and X∞ is the xanthine concentration at 48 h, were linear. This form of plotting is characteristic of a first-order (or pseudofirst-order) transformation which does not go to comple-

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 79

tion. The linear regression analysis of the data, while exhibiting excellent linearity as measured by R2, does not give slopes (rate constants) which are of value in determining relative reactivity, because the slopes of such plots are a function of X∞. Accordingly, we have calculated initial rates (Vo) from the derivative d[xanthine]/ dt ) -X∞me(mt+b) at time zero, where the derivative was determined from the equation X ) X∞[1 - e(mt+b)] of the exponential plot resulting from the regression analysis (above) where m is the slope of that plot and b is the intercept. The Vo values for xanthine formation from each of the nitrosamino aldehydes are given in Table 1 for both xanthine and hypoxanthine. Because of the lower levels of hypoxanthine formation, there is greater uncertainty in these values and at best the Vo values can be regarded as an indicator of relative reactivity. The data for xanthine show the reactivity order: NHMOR > BuNE . MeNE. In the case of hypoxanthine formation, there is a reversal in reactivity between BuNE and NHMOR, but this may be due to experimental error. In each case, the methyl compound is significantly less reactive. As was seen in the yield data, guanine deaminated more readily than adenine, and this is consistent with other observations on the relative rates of nitrosative deamination of DNA bases. We have considered various mechanisms for the transnitrosation reactions of R-nitrosamino aldehydes (6), which, in the case considered here, leads to deamination of DNA bases, but we have at present no definitive data which permit anything but pure speculation about how this very interesting transformation occurs. It is important to emphasize first that common dialkylnitrosamines do not enter into transnitrosation transformations except in very strong acid and second that under the neutral to basic conditions employed here, nitrite cannot enter into nitrosation processes which result in deamination. To deaminate nitrosamine and amines, nitrite must be converted into nitrous acid or oxides of nitrogen, a process which occurs only at pH values below 5. We do not yet know the transnitrosation mechanism of R-nitrosaminoaldehydes. Some of the N-nitroso groups may be converted into nitrite at physiological pH through “transnitrosation” to H2O, but it is unlikely that these ions are involved in the deamination chemistry. It is more probable that the nitroso group is transferred directly from the R-nitrosamino aldehyde or some derivative thereof to one of the purine or pyrimidine NH2 groups of DNA. The chemistry of R-nitrosaminoaldehydes is highly unusual in this context. Mechanism studies are underway. Through this investigation, it has become evident that R-nitrosaminoaldehydes, including NHMOR, the metabolite of NDELA, can deaminate DNA bases through transnitrosation. The transformations occur at concentration levels and on time scales which could be biologically significant. Deamination does not incorporate carbon fragments into the DNA backbone but can clearly produce mutations. This may explain why NDELA does not incorporate significant levels of its carbon fragments into DNA yet exerts strong carcinogenicity. In other work, we have shown that the in vivo administration of NDELA to rats results in the formation of gG and O6-(hydroxyethyl)guanine adducts (6, 20, 28).2 The former adducts 2 R. N. Loeppky and P. Goelzer, manuscript to be submitted for publication.

80

Chem. Res. Toxicol., Vol. 13, No. 2, 2000

Park and Loeppky

are hydrolytically unstable and may not have survived the analyses used to determine the extent of radiocarbon incorporation from NDELA into DNA. Our current data are consistent with bioactivation processes for NDELA which involve both its R- and β-oxidation (6, 7, 20), despite the fact that NHMOR has been reported to be only weakly carcinogenic (29). The latter experiments were carried out at very low doses, however. Clearly, NHMOR deaminates the three different types of bases in DNA. In vitro experiments have demonstrated that the β-oxidation of NDELA can be catalyzed not only by ADH but also by microsomes (6).3 Therefore, base deamination by NHMOR, in addition to adduct formation, must be considered as a possible mechanism of carcinogenesis for NDELA.

(10)

Summary and Conclusions

(11)

We have developed a convenient and simple method for the production of the reactive R-nitrosaminoaldehydes by the Dess-Martin oxidation of the corresponding alcohol. The deamination of the NH2-containing bases in nucleotides and DNA through the transnitrosation reaction from three different R-nitrosaminoaldehydes has been demonstrated. The methodology has utilized 32Plabeled nucleotides and quantitative analysis by means of the GC/MS-SIM method, employing a 15N-labeled xanthine. As expected, the extent of deamination depends on the structure and concentration of the R-nitrosaminoaldehyde, which are time dependent. The order of base deamination rates is consistent with other observations: guanine > adenine . cytosine. High concentrations of NHMOR deaminate guanine and adenine in calf thymus DNA up to 10.4 and 7.9%, respectively. Lower concentrations of NHMOR, closer to those generated in the metabolism of NDELA, to which humans are exposed, produced a significant amount of deamination in a short period of time. These results support a role for NHMORmediated base deamination in the mechanism of carcinogenicity of NDELA.

Acknowledgment. The support of this research by a grant (ES03953) from the National Institutes of Environmental Health Sciences is gratefully acknowledged. We also express our profound appreciation to the research group of Prof. Steven Tannenbaum for the sample of the 15N-labeled xanthine standard and discussion of the related analytical methodology.

(6) (7)

(8)

(9)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

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