Chem. Res. Toricol. 1995, 8, 617-624
617
Formation of N2-Tetrahydrofuranyl and N2-TetrahydropyranylAdducts in the Reactions of a-Acetoxy-N-nitrosopyrrolidine and a-Acetoxy-N-nitrosopiperidine with DNA Mingyao Wang, Ruth Young-Sciame, Fung-Lung Chung, and Stephen S. Hecht* American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received September 22, 1994@
We studied the reactions with DNA of a-acetoxy-N-nitrosopyrrolidine(a-acetoxyNPYR) and a-acetoxy-N-nitrosopiperidine (a-acetoxyNPIP) in order to obtain more information on adduct formation by metabolic activation via a-hydroxylation of two cyclic nitrosamines, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP). Enzyme hydrolysis and HPLC analysis of DNA t h a t had been reacted with unlabeled, [l4C1-, o r [3H]a-acetoxyNPYR permitted the positive identification of W-(tetrahydrofuran-2-yl)deoxyguanosine(THF-dG). It was identified by comparison of its W spectrum and retention time to those of a standard, by conversion upon NaBH4 treatment to W-(4-hydroxybutyl)deoxyguanosine, and by neutral thermal hydrolysis to 2-hydroxytetrahydrofuran (THF-OH). The levels of THF-dG in DNA exceeded t h a t of other adducts of a-acetoxyNPYR. Reaction of a-acetoxyNPIP with DNA followed by enzyme hydrolysis and HPLC analysis resulted in the positive identification of two diastereomers of W-(3,4,5,6-tetrahydro-W-pyran-2-yl)deoxyguanosine (THP-dG) by comparison of their retention times and W spectra to those of standards. The levels of THP-dG were similar to those of THF-dG formed from a-acetoxyNPYR. Neutral thermal hydrolysis of DNA that had been reacted with a-acetoxyNPIP produced 2-hydroxy-3,4,5,6-tetrahydro-W-pyran (THP-OH). Studies on the mechanism of formation of THF-dG and THP-dG indicated that stable cyclic oxonium ion-derived electrophiles could be their major precursors. Our data provide the first evidence for the formation of cyclic oxonium ion-derived DNA adducts from cyclic nitrosamines and indicate some potential differences in DNA binding between a-acetoxyNPYR and a-acetoxyNPIP. THF-OH and THP-OH released from DNA by neutral thermal hydrolysis may be useful as dosimeters of adduct formation by NPYR and NPIP.
Introduction
in Figure 1. Adducts are formed from open-chain intermediates such as diazonium ion 3, from cyclic oxonium N-Nitrosopyrrolidine (NPYR)l and N-nitrosopiperidine ions such as 4 or related electrophiles, and from enals (NPIP) are structurally related carcinogenic cyclic nisuch as 2-butenal (8). Adducts 5-7 and 12-14 have trosamines. Our interest in these compounds is based been characterized previously as products of the reaction on their differing carcinogenic activities in rats and their of a-acetoxyNPYR with calf thymus DNA (8,12). Three probable endogenous formation in humans, as detailed of these adducts-5, 6, and 12-are also formed in the in the accompanying paper (I). Since DNA adducts are hepatic DNA of rats treated with NPYR, and one of them, important in carcinogenesis and have potential utility 5 , has been detected in several other tissues as well (8as biomarkers for the uptake and metabolic activation 11). In the accompanying manuscript, we identified of carcinogens, we have studied their formation from aTHF-dG (11)as a product of the reaction of a-acetoxyacetoxy-N-nitrosopyrrolidine(a-acetoxyNPYR) and a-acNPYR with dG (1). In this study, we have analyzed DNA etoxy-N-nitrosopiperidine(a-acetoxyNPIP), which are reacted with a-acetoxyNPYR for the presence of THFstable precursors to the a-hydroxynitrosamines formed dG. In addition, we have employed labeled a-acetoxyin the metabolic activation of NPYR and NPIP. NPYR to further investigate the distribution of adducts Previous metabolism and mutagenicity studies as well formed from a-acetoxyNPYR and DNA. as structure-carcinogenicity investigations support the DNA adducts of NPIP have not been previously rehypothesis that a-hydroxylation of NPYR is its major ported, and only limited data are available on its metapathway of metabolic activation (2-13). The formation bolic activation. a-Hydroxylation has been established of adducts from a-hydroxylation of NPYR is illustrated as a metabolic process, but it is unclear a t present whether it is the major metabolic activation pathway of * To whom correspondence and requests for reprints should be NPIP (15-17). By analogy to NPYR, electrophilic interaddressed. mediates should be generated from a-hydroxyNPIP. This Abstract published in Advance ACS Abstracts, May 1, 1995. has been confirmed in two studies which have demonAbbreviations: NPYR, N-nitrosopyrrolidine; NPIP, N-nitrosopiperidine; a-acetoxyNPYFt, a-acetoxy-N-nitrosopyrrolidine;a-acetoxyNstrated the formation of dG adducts from a-acetoxyNPIP, PIP, a-acetoxy-N-nitrosopiperidine; dG, deoxyguanosine; THP-dG, W a s summarized in Figure 2 (1, 18). Two adducts-THP(3,4,5,6-tetrahydro-W-pyran-2-yl)dG;THF-dG, W-(tetrahydrofurandG (22) and 7-(2-oxopropyl)-1~-ethenodG(231-have 2-y1)dG THF-OH, 2-hydroxytetrahydrofuran (the cyclic and predominant form of 4-hydroxybutanal); THP-OH, 2-hydroxy-3,4,5,6-tetrahydro-W- been identified. In this study, we investigated the pyran (the cyclic and predominant form of 5-hydroxypentanal); 2,4presence of these adducts in DNA reacted with a-acetoxyDNP reagent, (2,4-dinitrophenyl)hydrazinereagent; NNN, N-nitrosoNPIP. nornicotine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone. @
0 1995 American Chemical Society 0893-228x/95/2708-0617~~9.~0l~
Wang et al.
618 Chem. Res. Toxicol., Vol. 8, No. 4,1995
I
N=O II
N=O
N=O
aceloryNPVR
NPYR 0
0
I
+
0-
THF-dG 11
8
bH
I' H 'od 12
13
14
OH
Figure 1. Adduct formation from a-acetoxyNPYR: a ) adducts 5-7 are detected by neutral thermal hydrolysis in reactions of a-acetoxyNPYR with dG and DNA (8, 12);5 and 6 have also been quantified in the livers of NPYR-treated rats (8, 10);(b) THF-dG (11) is detected i n reactions of a-acetoxyNPYR with dG ( 1 ) and DNA (this study); its origin is discussed in the text; (c) THF-dG is detected in reactions of 9 and 10 with dG (I) but not DNA (this study); (d) adduct 12 is detected in reactions of a-acetoxyNPYR or 2-butenal(8) with dG or DNA, followed by enzymatic hydrolysis (12-14). Only one of 2 diastereomers is illustrated. Adduct 12 has also been detected in the livers of rats treated with NPYR (9).Adducts 13 and 14 are detected by neutral thermal hydrolysis of dG reacted with a-acetoxyNPYR or 2-butenal (8).
II
N-0 acetoxyNPlP
e
19
o
HO +
n 20o = ( l O 21H
THP-dG
22
0
23
Figure 2. Adduct formation from a-acetoxyNPIP: ( a ) THP-dG (22)has been detected in reactions of a-acetoxyNPIP with dG (1) and DNA (this study). See text for discussion of its origin. Only one of 2 diastereomers is illustrated. (b) Adduct 23 has been detected in reactions of a-acetoxyNPIP with dG (18).Further details of the formation of 19-21 are given in refs 1 and 18.
Reactions of a-Acetoxynitrosamines with DNA
Experimental Section Caution. a-AcetoxyNPYR a n d a-acetoxyNPZP a r e mutagens a n d are chemically activated forms of the carcinogens NPYR a n d NPZP. They should be handled with extreme care, using appropriate safetywear a n d ventilation a t all times. Apparatus. HPLC analyses were carried out with Waters Associates (Millipore, Waters Division, Milford, MA) systems equipped with a Model 991 or 994 photodiode array detector, or a Perkin-Elmer Model 650-10s or LS-40 fluorescence detector, or a radioflow detector [Flo-one radioactive flow detector (Radiomatic Instruments, Meriden, CT) or P-RAM radioactivity HPLC detector (IN/US Systems, Inc., Tampa, FL)I. The following HPLC systems were used: (1) Two 3.9 mm x 30 cm pBondapak C-18 columns (Millipore, Waters Division) eluted isocratically with 20% MeOH in HzO at 1m u m i n with detection by W absorbance at 254 nm. (2) Two 4.0 mm x 25 cm Si-60 5 p m columns (E. Merck Lichrosorb, E. Merck Darmstaat, Germany) eluted isocratically with 60% CHC13 in hexane at 1 m u min with detection by UV absorbance at 254 nm. (3) Two 4.6 m m x 25 cm Partisil-10 SCX strong cation exchange columns (Whatman, Clifton, NJ) eluted with 0.05 M ammonium phosphate buffer (pH 2.01, at 1m u m i n , with fluorescence detection (excitation 290 nm; emission 380 nm). (4) A Protein Pak 5PW (diethy1amino)ethyl (DEAE) ion exchange column (Millipore, Waters Division) eluted with 5 M urea i n 20 mM sodium phosphate buffer (pH 6.9) without NaCl or with 1.5 M NaCl a s described (19). (5) A 4.6 mm x 25 cm Supelcosil LC 18-DB column (Supelco, Bellefonte, PA) eluted with a gradient from 0% to 30% CH3CN i n 10 mM phosphate buffer (pH 7.0), in 30 min at 1 m u m i n , using curve 6, with detection by W absorbance at 254 nm. (6) The same columns as in system 1 with a gradient from 50% to 100% MeOH in HzO in 50 min at 1 m u min, using curve 6. Detection was by UV abosrbance at 365 nm. (7) Two 3.9 mm x 30 cm yBondapak '2-18 columns (Millipore, Waters Division) eluted with 5% CH3CN in H2O for 5 min, then a linear gradient to 25% CH3CN in 30 min at 1 m u m i n . (8) A 4.6 mm x 25 cm Econosil 5 pm silica column (Alltech, Deerfield, IL) eluted isocratically with 7/3 CHCld hexane a t 1 m u m i n . Chemicals and Enzymes. a-AcetoxyNPYR, a-acetoxyNPIP, THF-OH, 4-hydroxybutanal (2,4-dinitrophenyl)hydrazone, 5-hydroxypentanal, 5-hydroxypentanal (2,4-dinitrophenyl)hydrazone, and standards of adducts 5-7, 11, 12-14, 22, and 23 were prepared as described (1, 8, 12-14, 18, 20-24). Preparations of the other chemicals are described below. DNase I (Type I1 from bovine pancreas) and phosphodiesterase I (Type VI1 from Crotalus atrox venom) were obtained from Sigma Chemical Co. (St. Louis, MO). Alkaline phosphatase (from calf intestine) was obtained from Boehringer Mannheim Corp. (Indianapolis, IN). [3H]a-AcetoxyNPYR. ~-[2,3,4,5-~HlProline (5 mCi; 102 Ci/ mmol) was obtained from Amersham Corp. (Arlington Heights, IL) as 1 mL of a n aqueous solution stabilized with 2%.ethanol. The solution was divided into two portions, and 0.4 mL of H2O and 0.1 mL of 1 N HC1 were added to each vial. Then, 20 mg (0.17 mmol) of unlabeled L-proline (Sigma Chemical Co.) and 14 mg (0.20 mmol) of NaNO2 were added, and the mixtures were heated at 37 "C for 1 h. The solvent was removed by rotary evaporation, and the residues were extracted with five 5 mL portions of CHzClz. The extracts were passed through a column packed with anhydrous MgS04. The CHzClz was removed by rotary evaporation, and the residue containing [3HW-nitrosoproline was redissolved in 1mL of dry CHzClz in a reaction vial filled with N2. To this were added 15 pL of dry pyridine and 100 mg (0.23 mmol) of lead tetraacetate (Aldrich Chemical Co., Milwaukee, WI) (20). The reaction mixture was heated under N2 at 40 "C overnight. The resulting mixture was purified on 20 cm x 20 cm silica gel F254s TLC plates (2 m m thickness) (EM Separations, Gibbstown, NJ) using CHCl&!H30H:100/1 as the eluting solvent. The band corresponding i n Rf to a-acetoxyNPYR was scraped into 50 mL of CH2C12. The extract was filtered, concentrated to dryness, and redissolved in 1mL of CH3OH for analysis and storage. The retention time of the [3H]a-
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 619 acetoxyNPYR was the same as that of unlabeled standard, 26 min, using reverse-phase HPLC (system 1). Three peaks were observed on normal-phase HPLC (system 21,eluting at 15, 16.1, and 18.2 min; the latter two coeluted with the E and 2 isomers of a-acetoxyNPYR. The purity of [3Hla-acetoxyNPYR used in these studies was approximately 90%. The yield of L3H]aacetoxyNPYR from ~-[2,3,4,5-3Hlprolineranged from 1.5% to 3.5% in different preparations. The specific activity ranged from 13.3 to 17.1 mCUmmo1. [14C]-a-AcetoxyNPYR(20 mCi"mo1) was prepared in similar fashion from ~-[UJ~C]proline (Amersham Corp.). This material was employed t o ensure that our results were not confounded by tritium exchange.
Reactions. (A) Reactions of a-AcetoxyNPYRand DNA. In some reactions, purified calf thymus DNA was used. Calf thymus DNA obtained from Sigma contained 0.2% protein. This was removed using (diethylaminolethy1 ion exchange HPLC, system 4 (19). The collected DNA was desalted on a Bio-Gel P6DG desalting column (Bio-Rad, Richmond, CA) followed by dialysis in HzO for 24 h. The resulting DNA solution was lyophilized and the DNA stored desiccated at -20 "C until use. Its purity was 99.9% by HPLC analysis, system 4. However, the results obtained with purified and unpurified DNA were the same, and in most reactions unpurified DNA was employed. DNA (10 mg) was dissolved in 2 mL of 0.1 M phosphate buffer (pH 7.0). This was added to [3Hla-acetoxyNPYR (30 mg, 0.19 mmol, 13.3 pCi, 0.07 mCi/mmol). The mixture was incubated at 37 "C for 21 h. The reaction mixture was extracted with 5 mL of CHCl&oamyl alcohol, 24/1, and centrifuged at 14000g for 20 min. The supernatant was removed, and 0.2 mL of 5 M NaCl and 4 mL of ethanol were added to precipitate the DNA. The DNA was washed with ethanol, ethanovether (VU, and ether, and then dried under a stream of N2. In some experiments, DNA (5 mg) that had been reacted with [3Hla-acetoxyNPYR was dissolved in 0.4 mL of 0.1 M phosphate buffer (pH 7.0) and further purified by HPLC system 5. It was then desalted and dialyzed as described above for calf thymus DNA. Results were unaffected by this additional purification. Reactions with [14Cla-acetoxyNPYRor unlabeled a-acetoxyNPYR were carried out a s described above. In the latter, 10 or 20 mg of DNA and 0.19 or 0.38 mmol of a-acetoxyNPYR were used. For enzyme hydrolysis, 5 mg of DNA was dissolved in 1.0 mL of 10 mM Tris-HCV5 mM MgClz buffer (pH 7.0), and the mixture was incubated with 2400 units of DNase I at 37 "C for 10 min, then with 0.15 units of phosphodiesterase I and 750 units of alkaline phosphatase for an additional 60 min. The enzymes were removed centrifugally using a n Amicon Centrifree Micropartition System (Amicon Division, W. R. Grace and Co., Danvers, MA). Control experiments demonstrated that these conditions resulted in complete hydrolysis of calf thymus DNA. The hydrolysates were analyzed using HPLC system 5. For neutral thermal hydrolysis, 3 mg of DNA was dissolved in 0.6 mL of 10 mM sodium cacodylate buffer (pH 7.4), and heated at 100 "C for 1 h. A 0.3 mL portion of the hydrolysate was treated with 30 pL of 1N HC1 to precipitate the DNA. The supernatant was analyzed for adducts 5,6,13, and 14 by HPLC system 3. The precipitated DNA was dissolved in 0.5 mL of 0.1 N HCl and heated at 80 "C for 45 min to release adduct 12 as the guanine base; it was analyzed by HPLC system 5. For analysis of 4-hydroxybutanal (2,4-dinitrophenyl)hydrazone,a 0.15 mL portion of neutral thermal hydrolysate was added to 10 mL of HzO, and then 0.1 mL of (2,4-dinitrophenyl)hydrazine reagent [2,4-DNP reagent (prepared from 125 mg of (2,4dinitropheny1)hydrazine in 50 mL of 6 N HCl)] was added. After 1h at room temperature, the mixture was extracted three times with 15 mL of CHzC12. The combined extracts were concentrated to dryness by rotary evaporation, and the residue was dissolved in 1mL of methanol for analysis by HPLC system 6. The retention time of 4-hydroxybutanal (2,4-dinitrophenyl)hydrazone was 23 min. For treatments with NaBH4, 0.3 mL of enzyme hydrolysate was treated with 40 pL of 1 M aqueous NaBH4 at room
620 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
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Wang et al.
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Figure 3. HPLC chromatograms (system 5) and LJV spectra (inserts) of (A) enzymatic hydrolysate of DNA that had been reacted with a-acetoxyNPYR and (B) the hydrolysate after treatment with NaBH4. Peaks corresponding to THF-dG, Wn-(4-hydroxybutyl)dG, and diastereomers of adduct 12 are indicated. UV spectra of the adducts are shown in the inserts. temperature for 2 h. The mixture was analyzed for W 4 4 hydroxybuty1)dG by HPLC system 5. (B) Reactions of Solvolysis Products of a-AcetoxyNPYR with DNA. a-AcetoxyNPYR (30 mg, 0.19 mmol) was incubated in 1 mL of 0.1 M phosphate buffer (pH 7.01, for 4.5 h at 37 "C. The resulting mixture was added to a solution of 10 mg of calf thymus DNA in 2 mL of 0.1 M phosphate buffer (pH 7.0) and heated a t 37 "C for 21 h. The DNA was enzyme hydrolyzed and analyzed for THF-dG using HPLC system 5 as described above. In a second experiment, a mixture of a-acetoxyNPYR (570 pmol) and 33 mg of DNA in 6 mL of 0.1 M phosphate buffer (pH 7.0) was incubated at 37 "C for 16 h. Then 6 mL of HzO was added and the mixture was extracted twice with 12 mL of CHCldisoamyl alcohol (24/1). The DNA was precipitated from the aqueous layer by addition of 1.2 mL of 5 M NaCl and 12 mL of ethanol. The organic extracts and aqueous layer were combined and concentrated to 1.2 mL. DNA, 33 mg in 6 mL of phosphate buffer, was incubated with 1 mL of this solution a t 37 "C for 16 h. The DNA was isolated, dissolved in 2 mL of 10 mM sodium cacodylate buffer (pH 7.41, and dialyzed for 36 h. It was then subjected to neutral thermal hydrolysis as above. The hydrolysates were treated with 2,4-DNP reagent and analyzed for 4-hydroxybutanal (2,4-dinitrophenyl)hydrazone using HPLC system 6. (C) Reaction of a-AcetoxyNPIP with DNA. a-hcetoxyNPIP (40 mg, 0.23 mmol) was added to a solution of 10 mg of calf thymus DNA in 2 mL of 0.1 M phosphate buffer (pH 7.0). The mixture was incubated at 37 "C in a shaking water bath for 24 h. Then 3 mL of HzO was added and the mixture was extracted twice with CHCldisoamyl alcohol (20/1). The DNA was precipitated with cold absolute EtOH and dried. The DNA was resuspended in 5 mL of 10 mM Tris buffer (pH 7.0) and incubated at 37 "C in a shaking water bath with DNase I ( 1 mg, 1700 units) for 10 min. Then phosphodiesterase I(0.15 unit) and alkaline phosphatase (450 units) were added, and the mixture was incubated for 60 min. The hydrolysates were concentrated by rotary evaporation, and the enzymes were removed centrifugally. The hydrolysates were analyzed using HPLC system 7. A second portion of 10 mg of DNA that had been reacted with a-acetoxyNPIP was resuspended in 1 mL of 0.1 M phosphate buffer (pH 7.0) and subjected to neutral thermal hydrolysis by heating for 1h a t 100 "C. The resulting mixture was extracted twice with 0.5 mL of CHC13. The CHC13 extracts were concentrated under a stream of Nz, and the residue was resuspended in 0.2 mL of H20 and derivatized with 10 pL of 0.13 M 2,4DNP reagent. The derivatized sample was extracted twice with 0.5 mL of CHC13, and the extracts were concentrated and
analyzed by HPLC system 8. The aqueous layer from above (0.25 mL) was treated with 50 pL of 0.13 M 2,4-DNP reagent. It was extracted with two 0.5 mL portions of CHC13, and the extracts were concentrated and analyzed for 5-hydroxypentanal (2,4-dinitrophenyl)hydrazoneby HPLC system 8. (D)Reactions of THF-OHand THP-OH with DNA. Calf thymus DNA (10 mg) was dissolved in 2 mL of 0.1 M phosphate buffer (pH 7.0). THF-OH (16.5 mg, 0.19 mmol) was added, and the mixture was incubated a t 37 "C for 20 h. DNA was isolated and analyzed for THF-dG a s described above. Reactions with THP-OH were carried out the same way with 10 mg of DNA and either 23.5 mg (0.23 mmol) or 6.8 mg (0.067 mmol) of THP-OH. The DNA was analyzed for THP-dG as described above.
Results Calf thymus DNA that had been allowed to react with unlabeled a-acetoxyNPYR was hydrolyzed enzymatically, and the hydrolysates were analyzed by HPLC for THFdG (11,Figure 1). The peak illustrated in Figure 3A had the same retention time and W spectrum as reference THF-dG. Treatment of the hydrolysate with NaBH4 caused this peak to shift to the retention time ofw-(4hydroxybutyl)dG, as shown in Figure 3B. The W spectrum of this peak was identical to that of a standard. The diastereomeric adducts 13,which are not affected by NaBH4 treatment under these conditions, can also be seen in Figure 3B. Neutral thermal hydrolysis of this DNA produced THF-OH, identified as its (2,4-dinitrophenyl)hydrazone, approximately 14 mmol/mol of dG. These data demonstrate the presence of THF-dG in enzyme hydrolysates of this DNA. Due to the instability of THF-dG, as described in the preceding paper, up to 70% of this adduct would be lost during enzymatic hydrolysis, and its level in DNA could only be estimated as 19 mmol/mol of dG. The reaction of a-acetoxyNPYR with DNA was further investigated using [3H]a-acetoxyNPYRor [l4C1a-acetoxyNPYR, which were synthesized from the appropriately labeled proline. Enzymatic hydrolysis of the reaction mixture obtained with [3Hla-acetoxyNPYR gave the chromatogram illustrated in Figure 4A. Reactions of [14C]a-acetoxyNPYRwith DNA gave similar results. The peak corresponding in retention time to THF-dG was one of the major peaks in the chromatogram. Its concentra-
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 621
Reactions of a-Acetoxynitrosamineswith DNA
q
I
dA
N
1: B-
/I
I -
THP-dG
I
> a
0 0
10
20
30 40 Time (min)
sb
6il
B
5 G
2
fE
0
9
27 Time (min)
18
36
45
Figure 4. HPLC chromatograms (system 5) of (A) enzymatic hydrolysate of DNA t h a t had been reacted with [3Hla-acetoxyNPYR and (B) the hydrolysate after treatment with NaBH4. The chromatograms in the upper panels of A and B were obtained with W detection while those in the lower panels were obtained with radioflow detection. The peaks corresponding in retention time to the normal deoxyribonucleosides, THF-dG, N 2 4 4 - h ~ droxybutyl)dG, and adduct 12 of Figure 1 are indicated.
tion was estimated as approximately 23 mmol/mol of dG. The peak eluting a t 11 min has not been identified. Treatment of the hydrolysate with NaBH4 gave the chromatogram shown in Figure 4B. The peak corresponding to THF-dG moved to the retention time of W (4-hydroxybutyl)dG,consistent with the results described above. The peak eluting at 11 min moved to 8.0 min, suggesting that it may be a THF-substituted derivative; this requires further investigation. Peaks corresponding to diastereomers of adduct 12 are also evident in Figure 4B. Neutral thermal hydrolysis of the enzymatic hydrolysis mixture resulted in disappearance of the peak corresponding to THF-dG. The peak eluting a t 11 min was unaffected by neutral thermal hydrolysis. Collectively, these data clearly demonstrate the presence of THF-dG as a major adduct in DNA reacted with a-acetoxyNPYR. While enzyme hydrolysis can be employed for detection of THF-dG and adduct 12 in this DNA, neutral thermal
10
20 Time (min)
30
40
Figure 5. HPLC chromatogram (system 7) of a n enzymatic hydrolysate of DNA t h a t had been reacted with a-acetoxyNPIP. Normal nucleosides and THP-dG diastereomers are indicated.
hydrolysis with fluorescence detection is the method of choice for detection and quantitation of the other adducts illustrated in Figure 1. Neutral thermal hydrolysis of DNA that had been reacted with [3Hla-acetoxyNPYRor [14C]a-acetoxyNPYRproduced both radioactive and fluorescent HPLC peaks corresponding in retention time (24 min) to adduct 5 of Figure 1; adducts 6,13,and 14 were detected only by fluorescence (data not shown). These Chromatograms also showed the presence of 2 peaks, eluting a t approximately 9 and 12 min, which accounted for 70-80% of the radioactivity in the chromatogram. The peak eluting a t 12 min had the same retention time as THF-OH, but its identity has not been confirmed. Levels of adducts formed in the reactions of a-acetoxyNPYR with DNA were as follows [adduct number from Figure 1,yield (mmol/mol of dG)]: THF-dG 11, 19; 5, 12.4; 6,6.6;12, 1.7; 13,0.2; 14,0.2. Enzyme-catalyzed hydrolysis of calf thymus DNA that had been allowed to react with a-acetoxyNPIP produced the chromatogram illustrated in Figure 5. The two peaks eluting a t 32.7 and 33.7 min had identical retention times to those of the two diastereomers of THP-dG (22,Figure 2). The UV spectra of these peaks were also identical to those of standard THP-dG. The amount of THP-dG produced in this reaction was 11.0 mmol/mol of dG. Neutral thermal hydrolysis of this DNA produced THPOH (13.4 mmol/mol of GI, identified as its (2,4-dinitropheny1)hydrazone derivative. A small peak corresponding in retention time to adduct 23 of Figure 2 was observed in the enzyme hydrolysates when UV detection a t 280 nm was employed, but its identity could not be confirmed by its UV spectrum. Since THF-OH and THP-OH react with dG, yielding THF-dG and THP-dG, respectively, their potential roles in the formation of these adducts in DNA was investigated. Reaction of THF-OH with DNA followed by enzymatic hydrolysis did not yield detectable amounts of THF-dG, in contrast to the results obtained in its reactions with dG. Reaction of THP-OH with DNA followed by enzymatic hydrolysis did produce THP-dG. Its presence was confirmed by the retention times of the two diastereomers which were identical to those of standard THP-dG and by their UV spectra. The concentration of THP-dG in this DNA was 0.5 mmoumol of dG compared to 11.0 mmoumol of dG in reactions of DNA with a n equimolar amount of a-acetoxyNPIP. In reac-
622 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
tions carried out starting with the amount of THP-OH that would be formed in the solvolysis of a-acetoxyNPIP, e.g., 29% of the amount used above, the level of THP-dG in DNA was accordingly lower, 0.14 mmoymol of dG. Collectively, these results indicate that THF-OH and THP-OH are not significant precursors to THF-dG and THP-dG in DNA reacted with a-acetoxyNPYR or a-acetoxyNPIP. The role of other solvolysis products of a-acetoxyNPYR in the formation of THF-dG was investigated. These studies were carried out by incubating a-acetoxyNPYR in pH 7.0 phospate buffer a t 37 "C for 4.5 h, conditions which result in complete hydrolysis of the a-acetoxynitrosamine. At the end of this 4.5 h period, DNA was added to the mixture and allowed to react for 21 h. Enzymatic hydrolysis and HPLC analysis of this DNA produced THF-dG; the yield was comparable to that formed in the direct reaction of a-acetoxyNPYR with DNA. In a second series of experiments, solvolysis products of a-acetoxyNPYR were incubated with DNA as above. Then the DNA was isolated and dialyzed to remove any unbound THF-OH. Neutral thermal hydrolysis of this DNA produced THF-OH, identified as its (2,4-dinitrophenyl)hydrazone.Collectively, the results of these experiments indicate that a stable product of the solvolysis of a-acetoxyNPYR reacts with DNA to yield THF-dG. This product is not THF-OH.
Discussion The results of this study provide definitive evidence for the presence of THF-dG and THP-dG in DNA reacted with a-acetoxyNPYR and a-acetoxyNPIP, respectively. While our previous studies using fluorescence detection indicated that adduct 5 of Figure 1 was the major one produced from a-acetoxyNPYR (121, the present results obtained with labeled a-acetoxyNPYR clearly show that this is not the case. These experiments indicate that THF-dG is one of the major DNA adducts produced in this reaction. Although quantitation of THF-dG is difficult due to its instability, our results suggest that it is present in higher concentration in DNA than any of the other identified adducts formed in the a-hydroxylation of NPYR. THP-dG is, to our knowledge, the first example of a DNA adduct formed from a-acetoxyNPIP. Neutral thermal hydrolysis of DNA that had been reacted with a-acetoxyNPYR or a-acetoxyNPIP released THF-OH and THP-OH, respectively. They are formed a t least partially from THF-dG and THP-dG. These compounds may be useful for quantifying DNA adduct formation in animals or humans exposed to NPYR or NPIP. They have functional groups which are suitable for derivatization to species that can be sensitively detected by combined gas chromatography-mass spectrometry, fluorescence, or other techniques. An additional advantage is that neutral thermal hydrolysis would separate the hydroxyalkanal analyte from the vast excess of unmodified DNA. We are presently investigating the use of (pentafluorobenzy1)hydroxylaminefor trace analysis of THF-OH and THP-OH released from DNA. Previously, we have employed a related electrophore derivatization approach to analyze 4-hydroxy-l-(3-pyridy1)-1-butanone released from DNA of animals or humans exposed to N-nitrosononicotine (NNN)or 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) (25). Three possible routes of formation of THF-dG are summarized in Figure 6. The same potential mecha-
Wang et al.
I N=O
4 1 3
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(070H
HO
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10
24
1
DNA path c
THF-dG THF-dG Figure 6. Three possible paths of formation of THF-dG from a-acetoxyNPYR and DNA (see text).
nisms apply to THP-dG. Path a involves direct reaction with the hydroxyalkanal. This is a n important pathway for production of THF-dG and THP-dG in reactions of the a-acetoxynitrosamines with dG, perhaps accounting for as much as 25% of the product, as discussed in the companion paper (I). However, we could not detect THFdG in reactions of THF-OH with DNA. Similarly, the yield of THP-dG in the reaction of THP-OH with DNA was relatively low, accounting for only about 2% of the THP-dG formed in the reaction of a-acetoxyNPIP with DNA. Therefore, path a does not appear to be important in the DNA reactions. The direct reaction of the cyclic oxonium ion 4 with DNA, path b, is a n attractive mechanism for production of THF-dG or, in the case of a-acetoxyNPIP, THP-dG. However, the relatively high yield of THF-dG in reactions that were carried out by addition of DNA after all the a-acetoxyNPYR had hydrolyzed argues against substantial involvement of this pathway because the oxonium ion is not expected to be stable. Therefore, the most likely mechanism of formation appears to be path c, in which the oxonium ion reacts either with buffer or with another solvolysis product to produce an oxonium ion-derived electrophile such as 24. This electrophile could then react with DNA to give THFdG or THP-dG. Experiments are in progress to identify this substance, which might be a phosphate or acetate derivative of THF-OH. We have made similar observations in our earlier studies on formation of adducts 5 and 6 in reactions of a-acetoxyNPYR with DNA (12). Kinetic studies indicated that both adducts were still forming up to 3 h after complete hydrolysis of a-acetoxyNPYR, indicating the presence of stabilized forms of intermediates such as 3 or 4 of Figure 1in the reaction mixture. Since path a of Figure 6 does not appear to be important in the formation of THF-dG and THP-dG in DNA, the results of these studies support the intermediacy of cyclic oxonium ions in the solvolysis of a-acetoxyNPYR and a-acetoxyNPIP. Paths b and c both require initial formation of a cyclic oxonium ion. In previous studies, we have obtained evidence for the involvement of cyclic oxonium ions in the a-hydroxylation of NNN and NNK (26). Cyclic oxonium ion formation in nitrosamine
Reactions of a-Acetoxynitrosamineswith DNA metabolism would be limited to bifunctional or cyclic nitrosamines. As in the reactions with dG, our results indicate some similarities and differences between the DNA binding products of a-acetoxyNPYR and a-acetoxyNPIP. Both nitrosamines form similar amounts of the cyclic oxonium ion-derived adducts THF-dG and THP-dG. Whereas products resulting from reaction of open-chain electrophiles such as 3 are quantitatively significant in DNA that has been reacted with a-acetoxyNPYR, we have not yet detected any such adducts in our studies of a-acetoxyNPIP. a-AcetoxyNPYR also forms enal derived adducts such a s 12-14 in DNA, whereas such adducts have not been detected from a-acetoxyNPIP. Our data indicate that low levels of 7-(2-oxopropyl)-l,W-ethenodG (23, Figure 2) are present in DNA reacted with a-acetoxyNPIP, but this requires confirmation with more sensitive methods. We have not detected ethenodG adducts in DNA reacted with a-acetoxyNPYR. The extent to which these differences in formation of adducts in vitro relates to the differing carcinogenic properties of a-acetoxyNPYR and a-acetoxyNPIP requires further in vivo studies. Little is known a t present about the formation in vivo, persistence, and biological significance of the adducts illustrated in Figures 1and 2. Adducts 5,6, and 12 have been detected in hepatic DNA of rodents treated with NPYR (8-11). The persistence of adduct 5 in rat, mouse, and hamster tissue DNA has been quantified, and there is some evidence for its longer persistence in two of the targets for NPYR carcinogenesis, rat liver and hamster lung (11). However, persistent high levels of adduct 5 have also been detected in nontarget tissues (11). Sitespecific mutagenesis studies on a n unsubstituted analogue of adduct 12,1,W-propanodG, have shown that it can cause G to T transversion mutations as well as frameshift mutations (27,28). The potential miscoding properties of THF-dG and THP-dG would depend partly on their stability in DNA. This is currently being assessed. Although THF-dG and THP-dG are unstable a t the nucleoside level, they may be more stable in DNA, as we have observed for adducts of NNN and NNK (29). It will be important to obtain more data on the in vivo stability and biological properties of THF-dG and THFdG. Since they modify the critical hydrogen bonding region of dG, it is likely that they will cause miscoding. In conclusion, the results of these studies have provided evidence for new modes of DNA adduction by cyclic nitrosamines involving the initial formation of cyclic oxonium ions and consequent reaction with the exocyclic amino group of dG in DNA. The release of THF-OH and THP-OH upon hydrolysis of this DNA provides a potential method for assessing the formation of such adducts in vivo.
Acknowledgment. This study was supported by Grant CA-44377 from the National Cancer Institute. This is paper 154 in “A Study of Chemical Carcinogenesis”.
References (1) Young-Sciame, R., Wang, M., Chung, F.-L., and Hecht, S. S.(1995) Reactions of a-acetoxy-N-nitrosopyrrolidineand a-acetoxy-N-
nitrosopiueridine with deoxyguanosine: formation of W-tetrahydrofuranyl or W-tetrahydriiyranyl adducts. Chem. Res. Toxicdl. 8 , 607-616. (2) Hecht, S. S., McCoy, G. D., Chen, C. B., and Hoffmann, D. (1981) The metabolism of cyclic nitrosamines. In N-Nitroso Compounds (Scanlan, R. A,, and Tannenbaum, S. R., Eds.) pp 49-75, American Chemical Society, Washington, DC.
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 623 (3) Lijinsky, W. (1992) Chemistry and Biology of N-Nitroso Compounds, pp 280-320, Cambridge University Press, Cambridge, England. (4) McCoy, G. D., Chen, C. B., Hecht, S. S., and McCoy, E. C. (1979) Enhanced metabolism and mutagenesis of nitrosopyrrolidine in liver fractions isolated from chronic ethanol-consuming hamsters. Cancer Res. 39, 793-796. (5) McCoy, G . D., Hecht, S. S., and McCoy, E. C. (1982) Comparison of microsomal inducer pretreatment on the in vitro a-hydroxylation and mutagenicity of N-nitrosopyrrolidine in rat and hamster liver. Environ. Mutagen. 4, 221-230. (6) McCoy, G. D., Hecht, S. S., Katayama, S., and Wynder, E. L. (1981) Differential effect of chronic ethanol consumption on the carcinogenicity of N-nitrosopyrrolidine and W-nitrosonornicotine in male Syrian golden hamsters. Cancer Res. 41, 2849-2854. (7) McCoy, G. D., and Koop, D. R. (1988) Reconstitution of rat liver microsomal N-nitrosophrolidine a-hydroxylase activity. Cancer Res. 48, 3987-3992. ( 8 ) Chung, F.-L., Wang, M., and Hecht, S. S. (1989) Detection of exocyclic guanine adducts in hydrolysates of hepatic DNA of rats treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with a-acetoxy-N-nitrosopyrrolidine. Cancer Res. 49,2034-2041. (9) Chung, F.-L., Young, R., and Hecht, S. S. (1989)Detection of cyclic 1,Wpropanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde. Carcinogenesis 10, 1291-1297. (10) Wang, M., Chung, F.-L., and Hecht, S. S. (1992) Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with Nnitrosopyrrolidine. Carcinogenesis 13, 1909-1911. (11) Hunt, E. J., and Shank, R. C. (1991) Formation and persistence of a DNA adduct in rodents treated with N-nitrosopyrrolidine. Carcinogenesis 12,571-575. (12) Wang, M., Chung, F.-L., and Hecht, S. S. (1989) Formation of acyclic and cyclic guanine adducts in DNA reacted with a-acetoxyN-nitrosopyrrolidine. Chem. Res. Toxicol. 2, 423-428. (13) Chung, F.-L., and Hecht, S. S.(1983) Formation of cyclic 1padducts by reaction of deoxyguanosine with a-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosamino)butanal,or crotonaldehyde. Cancer Res. 43, 1230-1235. (14) Chung, F.-L., Young, R., and Hecht, S. S.(1984) Formation of cyclic l,W-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (15) Leung, K.-H., Park, K. K., and Archer, M. C. (1978) a-Hydroxylation in the metabolism of N-nitrosopiperidine by rat liver microsomes: formation of 5-hydroxypentanal. Res. Commun. Chem. Pathol. Pharmacol. 19, 201-211. (16) Nakamura, M., Horiguchi, Y., and Kawabata, T. (1984) Effect of ascorbic acid and some reducing agents on N-nitrosopiperidine metabolism by liver microsomes. In N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer (O’Neill, I. K., von Borstel, R. C., Miller, C. T., Long, J., and Bartsch, H., Eds.) pp 547-552, International Agency for Research on Cancer, Lyon, France. (17) Rayman, M. P., and Challis, B. C. (1975) Oxidation of N nitrosopiperidine in the Udenfriend model system and its metabolism by rat-liver microsomes. Biochem. Pharmacol. 24,621626. (18) Hecht, S. S., Young-Sciame, R., and Chung, F.-L. (1992) Reaction of a-acetoxy-N-nitrosopiperidinewith deoxyguanosine: oxygen dependent formation of 4-oxo-2-pentenal and a 1,W-ethenodeoxyguanosine adduct. Chem. Res. Toxicol. 5, 706-712. (19) Murphy, S. E. (1989) DNA isolation from small tissue samples using anion-exchange HPLC. Carcinogenesis 10, 1435-1438. (20) Saavedra, J. E. (1979) Oxidation of nitrosamines. 1. Formation of N-nitrosoimminium ions through the oxidative decarboxylation of N-nitrosoproline, N-nitrosopipecolic acid, and N-nitrososarcosine. J. Org. Chem. 44, 4511-4515. (21) Lijinsky, W., Keefer, L., and Loo, J. (1970) The preparation and properties of some nitrosamino acids. Tetrahedron 26,5137-5153. (22) Hecht, S. S., Chen, C. B., and Hoffmann, D. (1978) Evidence for metabolic a-hydroxylation of N-nitrosopyrrolidine. Cancer Res. 38, 215-218. (23) Paul, R., and Tchelitcheff, S. (1948) Sur l’aldehyde y-hydroxybutyrique et ses derives (On 4-hydroxybutyraldehyde and its derivatives). Bull. SOC.Chim. Fr. 15, 197-203. (24) Hurd, C. D., and Saunders, W. H. (1952)Ring-chain tautomerism of hydroxy aldehydes. J . Am. Chem. SOC.14, 5324-5329. (25) Foiles, P. G., Akerkar, S. A,, Carmella, S. G., Kagan, M., Stoner, G. D., Resau, J. H., and Hecht, S. S.(1991) Mass spectrometric analysis of tobacco-specificnitrosamine-DNA adducts in smokers and nonsmokers. Chem. Res. Toxicol. 4, 364-368. (26) Spratt, T. E., Peterson, L. A,, Confer, W. L., and Hecht, S. S. (1990) Solvolysis of model compounds for a-hydroxylation of W-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone:
624 Chem. Res. Toxicol., Vol. 8, No. 4, 1995 evidence for a cyclic oxonium ion intermediate in the alkylation of nucleophiles. Chem. Res. Toxicol. 3, 350-356. (27) Grollman, A. P. (1989) Molecular mechanisms of chemical mutagenesis. Proc. Am. Assoc. Cancer Res. 30,682. (28) Benamira, M., Singh, U., and Marnett, L. J. (1992) Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG14 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector. J . Biol. Chem. 267, 2239222400.
Wang et al. (29) Hecht, S. S., Peterson, L. A., and Spratt, T. E. (1994) Tobaccospecific nitrosamines. In DNA Adducts: Identification and Biological Significance (Hemminki, K., Dipple, A,, Shuker, D. E. G., Kadlubar, F. F., Segerback, D., and Bartsch, H., Eds.) IARC Scientific Publications No. 125, pp 91-106, International Agency for Research on Cancer, Lyon, France.
TX9401270
Announcements International Congress on Free Radicals in Health and Disease The Turkish Biochemical Society (Istanbul Branch) and the Society for Free Radical and Antioxidant Research (Istanbul) are organizing the International Congress on Free Radicals in Health and Disease, September 6-10,1995, a t the Holiday Inn Crowne Plaza in Istanbul, Turkey. The Congress is sponsored by the UNESCO Molecular and Cell Biology Network, The International Union of Biochemistry and Molecular Biology, and the Society for Free Radical Research (Europe). The Congress will provide a survey on the present state of knowledge and future trends in free radical and antioxidant research. The scientific program will be multidisciplinary and include invited lectures, oral presentations, and poster presentations on a wide range of issues such as basic concepts, methodology, nutrition, cardiovascular disease, cancer, antioxidants, and inflammatory and nervous system disorders. For further information, please contact: Congress Secretariat, MEDI Organization and Tourism Investments Inc., MBE P.K. 150 Dolapdere Cad. No: 283-287,80260 Pangalti, Istanbul, Turkey (telephone: (90) 212-246 24 15; (90) 212-246 13 92; or (90) 212-240 48 78; telefax: (90) 212-246 62 23). TX950475H
2nd World Congress on Alternatives and Animal Use in the Life Sciences The 2nd World Congress on Alternatives and Animal Use in the Life Sciences will be held October 20-24, 1996, in Utrecht, The Netherlands. The aim of the congress is to promote exchange of information on recent developments in the field of alternatives to animal use and, in particular, to review the progress that has been made in this respect within the fields of biomedical research, testing, and education. Platform sessions and workshop discussions will be organized to facilitate an objective assessment of the alternatives and their status. The Congress also intends to contribute to ongoing dialogue among the animal protection movement, the scientific community, regulatory authorities, and industry. Topics that will be covered include: Alternatives in (1)Basic Research; (2) Pharmacology; (3) Toxicology; (4) Vaccine Testing; and (5) Biologicals; ValidatiodRegulations; Animal Welfaremthics; and EducatiodDatabases. For further information, please contact World Congress Alternatives 1996, FBU Congress Bureau, P.O. Box 80.125,3508 TC Utretcht, The Netherlands (phone: +31 30 53 5044/2728; fax: +31 30 53 3667; Email:
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Meeting Calendar July 23-27, 1995
9th International Conference on Cytochrome P450 [Chem.Res. Toxicol. 7 (51, 701, 19941.
August 27-31, 1995
Fourth International ISSX Meeting [Chem.Res. Toxicol. 8 (3),477,19951.
October 11-13, 1995
European Conference on Combination Toxicology [Chem.Res. Toxicol. 8 (21, 321, 19951.
TX9504725
