Formation of NADP (H) Analogs of Tobacco-Specific Nitrosamines in

Mar 7, 1994 - Toxicol. 1994, 7, 599-608. 599. Formation of NADP(H) Analogs of Tobacco-Specific. Nitrosamines in Rat Liver and Pancreatic Microsomes1^...
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Chem. Res. Toxicol. 1994,7, 599-608

599

Formation of NADP(H)Analogs of Tobacco-Specific Nitrosamines in Rat Liver and Pancreatic Microsomes? Lisa A. Peterson,*'$Dennis K. Ng,$ Ralph A. Steams,$and Stephen S. Hecht$ American Health Foundation, 1 Dana Road, Valhalla, New York 10595, and Department of Drug Metabolism, Merck Research Laboratory, Rahway, New Jersey 07065 Received March 7, 1994@

The metabolism of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone (NNK), and its metabolite 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanol (NNAL), was examined in rat pancreatic microsomes. No pyridine N-oxidation or a-hydroxylation were observed in these preparations. However, one unidentified metabolite of NNAL (unknown A) and two unknown metabolites of NNK (unknowns B and C) were formed. These metabolites were also detected in rat liver microsomal incubations of NNK and NNAL. Studies using [5-3HlNNK and [Me-3H]NNK demonstrated that the metabolites contained both the pyridyl and methyl portions of the parent compound. Similar results were obtained with NNAL. Formation of unknown C required active microsomes, NADP+, and an NADPH regenerating system. The regenerating system was not required for the formation of NNAL unknown metabolite A or NNK unknown metabolite B. Chemical characterization of unknowns A and B by NMR, W, and electrospray ionization MS demonstrated that they are NADP+ analogs in which the nicotinamide portion has been replaced by NNAL or NNK, 4-(methylnitrosamino)-l-(3-pyridyl)1-butanol adenosine dinucleotide phosphate [(NNAL)ADP+l and 4-(methylnitrosamino)-1-(3pyridy1)-1-butanone adenosine dinucleotide phosphate [(NNK)ADP+l. Unknown C was identified as (NNK)ADPH. Both (NNK)ADP+ and (NNKIADPH were formed from NNK while only (NNAL)ADP+ was produced from NNAL. These NADP+ derivatives were also formed when porcine brain NAD+ glycohydrolase was incubated with NADP+ and NNK or NNAL. These results indicate that NNK and NNAL are substrates for rat liver and pancreatic microsomal NAD' glycohydrolase-catalyzedtransglycosylation reactions.

Introduction The tobacco-specificcarcinogen 4-(methy1nitrosamino)1-(3-pyridyl)-l-butanone(NNK)' primarily induces lung tumors in laboratory animals (1-6). Other sites of tumor formation include nasal cavity, trachea, liver, and pancreas. The pancreatic tumors are induced in F344 rats when NNK and its metabolite 4-(methylnitrosamino)-l(3-pyridyl)-l-butanol (NNAL) are given in the drinking water (6, 7). The combination of ethanol and NNK is a potent transplacental inducer of pancreatic tumors in hamsters (8). Since smoking is associated with an increased risk of pancreatic cancer in humans (9), it is of interest to explore the mechanism of pancreatic tumor induction by these nitrosamines. + This is Paper 155 of the series "A Study of Chemical Carcinogenesis". *To whom correspondence and reprint requests should be addressed. American Health Foundation. 5 Merck Research Laboratory. @Abstractpublished in Advance ACS Abstracts, July 15, 1994. Abbreviations: NNK, +(methylnitrosamino)-l-( 3-pyridyl)-l-butanone; NNAL, 4-(methylmtrosamino)-l-(3-pyridyl)-l-butanol; keto aldehyde, 4-oxo-l-(3-pyridyl)-l-butanone; keto alcohol, 4-hydroxy-l-(3pyridy1)-1-butanone; keto acid, 4-oxo-4-(3-pyridyl)butyricacid; diol, 4-hydroxy-l-(3-pyridyl)-l-butanol; lactol, 2-hydroxy-5-(3-pyridyl)tetrahydrofuran; hydroxy acid, 4-hydroxy-l-(3-pyridyl)butyricacid; NNKN-oxide, 4-(methylnitrosamino)-l-(N-oxy-3-pyridyl~-l-butanone; NNALN-oxide, 4-(methylnitrosamino)-l-(N-oxy-3-pyridyl)-l-butanol; NADP+, nicotinamide adenosine dinucleotide phosphate, PMSF, phenylmethanesulfonyl fluoride; (NNKIADP', 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone adenosine dinucleotide phosphate; (NNAL)ADP+, 4-(methylnitrosamino)-l~3-pyridyl)-l-butanol adenosine dinucleotide phosphate; ADPR, adenosine dinucleotide phosphate ribose; BOP, N-nitrosobis(2-oxopropy1)amine; HPOP, N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine. Enzyme: NAD(P) nucleosidase or NAD(P) glycohydrolase (EC 3.2.2.6).

*

Q893-228x/94I27Q7-Q599$04.5QlQ

NNK is believed to require metabolic activation to elicit its carcinogenic effects. The principle pathways of metabolism, as outlined in Figure 1, include a-carbon hydroxylation, N-oxidation, and carbonyl reduction to NNAL ( 1 0 , l l ) . N-Oxidation of NNK is thought to be a detoxification route (2). The a-carbon hydroxylation routes activate NNK to either a DNA methylating or pyridyloxobutylating species (12,131. The primary products of these two pathways have been detected in microsomal incubations of NNK as 4-oxo-l-(3-pyridyl)1-butanone (keto aldehyde) and 4-hydroxy-l-(3-pyridyl)1-butanone (keto alcohol), respectively (14-17). The observation that microsomes from a variety of tissues catalyze the formation of these two compounds indicates that these tissues are capable of activating NNK t o a DNA reactive species (18). The ability of pancreatic microsomes to activate NNK via a-carbon hydroxylation has not been previously investigated. Reduction of NNK to NNAL does not remove the risk of tumor formation. NNAL also induces lung and pancreatic tumors in laboratory animals (2,6, 7, 19). This metabolite is a carcinogen either via a-hydroxylation or by oxidation to NNK followed by a-hydroxylation (18). The primary products of a-hydroxylation of NNAL have not been characterized in microsomal incubations. The expected products of NNAL a-hydroxylation are 4-hydroxy-l-(3-pyridyl)-l-butanol(diol) and 2-hydroxy-543pyridy1)-tetrahydrofuran(lactol, Figure 1).Diol has been observed as a metabolite of NNAL cultured with mouse peripheral lung (2). Secondary products such as 4-hydroxy-l-(3-pyridyl)butyricacid (hydroxy acid) have been detected in tissue culture incubations of NNAL (2,20). NNAL-N-oxide is formed when NNK is incubated with

0 1994 American Chemical Society

600 Chem. Res. Toxicol., Vol. 7, No. 5, 1994

Peterson et al. COOH

+

NNK NNK.N-oride

I

m" 1

+ ICH,N=NOH]

N

0

helo aldehyde

[

I q N = N . O H ]

1

+HCHO

CHiOH

c OH

Figure 1. Metabolic pathways for NNK and NNAL.

microsomes or cultured tissue (21-23). 0-Glucuronides of NNAL are also formed in vivo (24-26). In the current study we determined the ability of rat pancreatic microsomes to metabolize NNK and NNAL. While they were unable to catalyze a-carbon hydroxylation or N-oxidation, one unknown metabolite of NNAL (unknown A) and two unknowns from NNK (unknowns B and C) were generated. These compounds were also observed in rat liver microsomal incubations of NNK and NNAL. Chemical characterization demonstrated that they were NADP(H) analogs in which nicotinamide had been replaced by NNAL or NNK.

Experimental Section Caution: NNK and NNAL are carcinogens and must be handled with care. Materials. Male F344 rats (200-400 g) were purchased from Charles River Laboratories (Kingston, NY). Some of the pancreatic microsomes were prepared from male F344 r a t pancreas purchased from Harlan Bioproducts (Indianapolis, IN). NNK and NNAL were synthesized according to published procedures (10, 27). [5-3HlNNK (specific activity 1.8, 2.51, or 2.89 CUmmol) and [Me-3HlNNK(specific activity 1.06 Ci/mmol) were purchased from Chemsyn Laboratories (Lenexa, KS). [5-3HlNNAL and [Me-3HlNNAL were prepared from the corAU responding r3H1NNK according to published procedures (10). radiochemicals were purified by C18 chromatography using HPLC method A (see below). Glucose 6-phosphate, glucose-6phosphate dehydrogenase, NADP+, NADPH, NAD+, phenylmethanesulfonyl fluoride (PMSF), pepstatin, and porcine brain NAD+ glycohydrolase were purchased from Sigma Chemical Co. (St. Louis, MO). Instrumental Analyses. lH NMR spectra were acquired with a Bruker Model AM 360 WB spectrometer and are reported in ppm relative to an external standard (tetramethylsilane, DzO). The unCDC13; 2,2-dimethyl-2-silapentanesulfonate, knowns A and B and NADP+ were analyzed on a SCIEX API I11 tandem mass spectrometer. Ionization was induced by ion spray (pneumatically assisted electrospray). U V spectra were recorded with a Beckman UV spectrometer. HPLC analyses were performed with a Waters 510 system (Millipore, Waters

Division, Milford, MA), and radiochemical detection was performed by either FLO-onemeta (Radiomatics Instruments, Tampa, FL) or Beta-ram (Inus Systems Inc, Tampa, FL) radiochemical detectors. Some HPLC analyses were performed with a Waters 991 photodiode-array detector. HPLC Methods. Several different chromatographic systems were used in these studies. Method A utilized solvent A (20 mM sodium phosphate buffer, pH 7) and solvent B (95% methanol, 5% HzO). Radiolabeled NNK and NNAL were purified on a C18 column (Bondaclone 10, 30 x 0.39 cm, Phenomenex, Torrance, CA) using a linear gradient from 100% solvent A to 92% solvent A over 16 min. After 15 min another linear gradient was run to 67% A over 50 min. Method B used the same solvents. However, the metabolites were eluted with a linear gradient from 100% solvent A to 65% solvent A over 60 min. In this system, 4-(methylnitrosamino)l-(3-pyridyl)-l-butanol adenosine dinucleotide phosphate [(NNAL)ADP+]and 4-(methylnitrosamino)-l-(3-pyridyl)-l-b~tanone adenosine dinucleotide phosphate [(NNK)ADP+lelute at approximately 18-20 min and (NNK)ADPH elutes a t 24-26 min. Method C uses the same solvents with a linear gradient from 100% solvent A to 50% solvent A over 40 min. (NNK)ADP+and (NNAL)ADP+now elute a t 16 min and (NNKIADPH elutes a t 19.5 min. Method D elutes the metabolites from the same column with solvent C (20 mM sodium phosphate buffer, pH 6) and solvent B using the same linear gradient as in method B. In this system, the NADP+ analogs have approximate retention times of 22-24 min and (NNK)ADPH elutes a t 30-32 min. Preparative isolation of the NADP+ derivatives was achieved using the three following HPLC methods. Method E involves a linear gradient from 100% solvent A to 85% solvent A/15% solvent B over 30 min, followed by a 15-min gradient to 50% solvent A/50% solvent B. In method F the metabolites are separated with a linear gradient from 100% solvent C to 85% solvent C (15% B) over 30 min followed by a 5-min gradient to 80% solvent C (20% B). Method G utilizes solvent D (10mM ammonium acetate) and solvent B with a linear gradient from 100% D to 80% D over 20 min. Microsomal Preparations. Liver microsomal fractions were prepared by the method of Guengerich et al. (28). Pancreatic microsomal preparations were obtained using the method

NADP(H) Analogs of NNK and NNAL in Microsomes

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 601

Table 1. 'H NMR Spectra of NADP+,(NNK)ADP+,(NNAL)ADP+,NNK, and NNALa chemical shifts (ppm) N=O

R

I

NADP+ PyridYl 2 4 5 6 butyl 1 2 3 4 (ON)NCH3 ribose 1' 1"

2' 3',4',5',2,3",4",5" adenine 2 8

9.35 (9) 8.85 (d) 8.25 (dd) 9.15 (d)

I R

R

(NNK)ADP+ (unknown B)

protons

N=C

9.3 ( 8 ) 8.8 (d) 8.15 (dd) 9.1 (d)

(NNAL)ADP+ (unknown A) 8.85 ( 8 ) 8.4 (dd) 7.95 (dd) 8.8 (d)

NNAL

NNK

9.1 ( 8 ) 8.1(dd) 7.3 (d) 8.65 (d)

8.6 ( 8 ) 7.7 (d) 7.3 (dd) 8.55 (d)

-b -c

E,2.15; Z,1.95 (t) E,4.2; Z,3.8 (t) E,3.15; 2,3.8 ( 8 )

E , 4.8; Z,4.75 (t)

1.9 (m) 1.7 (m) E, 4.25; Z,3.8 (m) E, 3.1; Z 3.7 (9)

6.2 (d) 6.1 (d) 5.1 (m) 4.2-4.gd

6.05 (d) 6.05 (d) 4.1-4.Bd

5.9 (d) 6.1 (d) 5.1 (m) 4.1-4.7d

8.2 (s) 8.45 (9)

8.1 (s) 8.35 (s)

8.1 (s) 8.4 (s)

-b

E,3.0; Z,2.9 (t) E,2.2; Z,1.9 (m)

1.9-2.5 (m) 1.9-2.5 (m) E, 4.2; Z,3.6 (m) E,3.0; 2,3.7 ( 8 )

E, 4.2; Z,3.6 (t) E, 3.0; Z,3.7 (s)

a NMR spectra were taken in D2O [NADP+,(NNK)ADP+,and (NNAL)ADP+] and CDCl3 (NNK and NNAL). Signals are reported in ppm relative to an external standard. s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets. Signal was obscured by HDO signal. Protons had been exchanged with D2O. Signals appeared as a series of multiplets in this region.

of Flammang et al. (29)with the addition of 5 pg/mL pepstatin to the washing and homogenizing buffers. Assays. Initial studies involved duplicate incubations of [5-3HlNNK,[5-3HlNNAL,[MeJHINNK, or [Me-3HlNNAL(1pM) with pancreatic or liver microsomes (1 mg/mL) in the presence of 100 mM potassium phosphate buffer (pH 7.4), 3 mM MgC12, 1 mM EDTA, 5 mM NADP+, 25 mM glucose 6-phosphate, and 1unit of glucose-6-phosphate dehydrogenase; (total volume 0.5 mL). Controls were performed with boiled microsomes or in the absence of NADP+, microsomes, or the regenerating system. Several incubations were conducted with 5 mM NAD+. Incubations were 1 h at 37 "C. The reactions were terminated by the addition of 0.3 N barium hydroxide and 0.3 N zinc sulfate (50 pL each) prior to cooling on ice. The mixture was filtered through an Acrodisc (Gelman, Ann Arbor, MI; 0.45 pm, 3 mm) and analyzed directly by reverse-phase HPLC linked t o radiochemical detection using HPLC methods B, C, or D.

Preparative Isolation of the Unknown Metabolites. Unknowns A and B were isolated from separate 50-mL incubations of [5-3H1NNAL(5 mM, 6.4 pCi) or [ 5 J H ] " K (5 mM, 42.5 pCi) with rat liver microsomes (1mg/mL) and NADP+ (5 mM) in 0.1 M potassium phosphate buffer (pH 7.4) containing 1mM EDTA and 3 mM MgC12. After 1 h a t 37 "C, the reactions were stopped by the addition of 0.3 N barium hydroxide and 0.3 N zinc sulfate (5 mL each). The supernatants obtained following centrifugation were extracted five times with methylene chloride (50 mL). The methylene chloride layer was discarded, and NNK unknown B was isolated from the aqueous phase using HPLC method E. Unknown B eluted a t approximately 19 min. This fraction was then further purified using HPLC method F in which the unknown metabolite eluted a t 22 min. This fraction was collected, desalted using a C18 Sep pak (Waters, Milford, MA), and concentrated. The NMR spectrum of this compound was acquired in DzO (Table 1).

Table 2. Molecular Ions of NADP+,(NNK)ADP+ (Unknown B). and Ol"N)ADP+ (UnknownA)" mlz

M+ NADP+ NNK(ADP)+ NNAL(ADP)+

744 829 831

(M

+ Na)+ 766 851 853

(M

+ 2Na)+ 788 873 875

a Determined by electrospray ionization mass spectral analysis. Samples (approximately 20 pg) were dissolved in HzO (200 pL) and analyzed by flow injection (5-10 pL; flow rate 40 pUmin). The mobile phase consisted of 50% CHsCN-10 mM NH40Ac0.1% TFA. Spectra were obtained by scanning from 350 to 900 Da with a dwell time of 2 malDa.

NNAL unknown A was purified in a similar manner. However, in this case, the initial purification was performed using HPLC method F first followed by repurification using HPLC method E. The retention times for this unknown were similar to that of NNK unknown B. This fraction was collected and desalted by C18 chromatography eluting with 100% H2O. The NMR spectrum of this metabolite was obtained in D2O (Table 1). Unknowns A and B were subjected to further C18 chromatography using method G. The corresponding peaks were collected and organics removed and lyophilized. For electrospray mass spectral analysis, samples (approximately 20 pg) were dissolved in 200 ,uL of HzO, and 5-10 pL was analyzed by flow injection (40 pL/min). The mobile phase consisted of 50% CH3CN-10 mM NH40Ac-O.l% TFA. Spectra were obtained by scanning from 350 to 900 Da with a dwell time of 2 ms/Da. The molecular ions of the metabolites and their mono- and disodium adducts are presented in Table 2. Product ion spectra were obtained from the molecular ion of each compound. For these experiments, the collision gas was argon and the collision energy was 30 eV. Spectra were obtained by scanning from 10 to 850 Da with a dwell time of 2 ms/Da.

Peterson et al.

602 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 In order to confirm that the unknowns observed in pancreatic microsomal incubations were similar to those observed in liver microsomal incubations, semipreparative incubations of [5-3H]NNK or [5-3H]NNAL (1 mM, 14 pCi) with pancreatic microsomes (1 mg/mL) and required cofactors were performed a s described above (totalvolume 14 mL). Purification of unknowns A-C was achieved by HPLC chromatography. First, the mixtures were separated on a C18 column using HPLC method

E. The radioactive fraction from the NNAL incubation (18-22 min) was then rechromatographed on the same column using HPLC method F. Unknown A eluted a t 24 min. This sample was desalted using a C18 Sep pak (Millipore, Waters Division, Milford, MA). After concentration, the W spectrum was obtained in methanol-& (Am, 259 nm). During the initial purification of the [5-3HlNNK incubation mixture, a fraction containing both NNK unknowns B and C was collected (20-30 min). This fraction contained two radioactive peaks that eluted at 22 and 28 min when the C18 column was eluted with a linear gradient from 100% C to 75% C over 30 min. Insufficient amounts of the 22-min NNK peak (unknown B) were obtained. Fractions containing unknown C were desalted on the C18 column using isocratic 85% Hz0/15% methanol. W and NMR spectra of this metabolite were obtained in methanol-& (Am, 365 nm). In order to characterize the other metabolites of NNAL generated in the liver microsomal incubations, a 10-mL incubation of 2 mM NNAL (8 pCi) with r a t liver microsomes (1 mg/ mL), NADP+, and the regenerating system (see above) was performed for 60 min a t 37 "C. Following protein precipitation with barium hydroxide and zinc sulfate, the supernatant was desalted by passage over a C18 Sep pak. NNAL and its metabolites were eluted with methanol. This fraction was concentrated to dryness under a nitrogen stream and resuspended in ethyl acetate. This solution was then passed over a silica Sep pak. The eluent and subsequent ethyl acetatemethanol (7:3) wash were combined and concentrated under reduced pressure. The residue was dissolved in HzO (2 mL). Analysis by C18 chromatography linked to radiochemical detection (method C) showed radioactive peaks corresponding to all NNAL metabolites with the exception of (NNAL)ADP+. This solution was then submitted for LC-MS analysis on a Fisons Instruments (VG) Quattro instrument by electrospray ionization in the positive ion CI mode. The metabolites were eluted from a 25 cm x 4.6 mm 5-pm Whatman (Clifton, NJ) Partisphere C18 column with 10 mM ammonium acetate using the following methanol gradient: a linear gradient from 0% t o 7% methanol over 7 min; then the system was held isocratic for 15 min followed by a 28-min gradient from 7% methanol to 35% methanol (flow, 1 mumin). Standard NNK metabolites eluted under these conditions as follows: keto acid, 9.6 min; NNAL-N-oxide, 19.2 and 20.9 min; diol, 27.4 min, NNK-N-oxide, 29.5 and 30.5 min; NNAL, 40.2 and 41.1 min. Analysis of this mixture was also performed on a C18 column linked to a diode-array detector. The column was eluted using HPLC method B. Standards of NNAL-N-oxide, diol, and keto alcohol (lactol) eluted a t 24.7 and 26, 28, and 38.5 min, respectively. UV spectra of the unknowns were taken and matched against standards for the predicted NNAL metabolites: NNAL-N-oxide, A,= 211 and 255 nm; diol, A,,,= 200 and 260 nm; and lactol, A,, 210,235, and 260 nm.

Formation of (NNK)ADP+ and (NNAL)ADP+by NAD Glycohydrolase. Porcine brain NAD+ glycohydrolase (1mg/ mL) was incubated with 1pM [5-3HlNNK or [5-3HlNNALand NADP+ (5 mM) i n 100 mM potassium phosphate buffer a t 37 "C for 60 min (total volume 0.5 mL). The reaction was stopped by the addition of 0.3 N barium hydroxide and 0.3 N zinc sulfate (50 pL each). After centrifugation and filtration, the samples were analyzed using HPLC method D.

Stability of the Unknown Metabolites to Acid, Base, or Neutral Thermal Hydrolysis. Fractions containing the unknown metabolites from liver and pancreatic microsomal or NAD glycohydrolase incubations (generated as described above

using 1 pM [5-3HlNNK or [5-3HlNNAL) were collected using HPLC method B or C. Portions of these fractions were submitted to 0.1 N NaOH, 0.1 N HCl, or neutral thermal hydrolysis a t 80 "C for 30 min. Following neutralization, the fractions were analyzed using HPLC method C linked to radiochemical detection.

Concentration Dependence of NNK and NNAL Metabolite Formation. [5-3H]NNK (1-3000 pM, 1.4 pCi) or [EL3H1NNAL (1-2000 pM, 1.55 pCi) was incubated with r a t liver microsomes (1mg/mL) and NADP+ (total volume 0.5 mL) for 60 min a t 37 "C. The NNAL. incubations were performed in the presence of the NADPH regenerating system (see above). The NNK incubations were analyzed using HPLC method C, and the NNAL incubations were analyzed using HPLC method

B.

Results and Discussion Incubations of 1pM [5-3HlNNALor [C~-~H]NNK with rat pancreatic microsomes (1mg of proteidml) in the presence of NADP+ and an NADPH regenerating system produced one unknown radioactive metabolite from NNAL (peak A, Figure 2a) and two unknown radioactive metabolites from NNK (peaks B and C, Figure 2B). These compounds did not coelute with any of the NNK metabolite standards illustrated in Figure 1,demonstrating that rat pancreatic microsomes were unable to catalyze a-hydroxylation or N-oxidation reactions under these conditions. However, they were able to support the reduction of NNK to NNAL. The formation of the unknown metabolites required NADP+ and active microsomes. Substitution of NADPH for NADP+ reduced the levels of unknowns formed. In the NNK incubations, the relative amounts of unknowns B and C varied within duplicates, but the total remained constant. When the NADPH regenerating system (glucose-6-phosphate dehydrogenase and glucose 6-phosphate) was omitted from the NNK incubation mixtures, only unknown B was observed (Figure 3A). NNAL unknown A and NNK unknown B coelute with one another. In order to determine which portion of the parent compound was retained in the unknown metabolites, incubations were performed with either 1pM L3H1NNK or [3H]NNALlabeled either at the 5-pyridyl position or in the methyl group. The results from these experiments indicated that the new unknowns formed in the pancreatic microsomal incubations of NNK and NNAL contained both the methyl and pyridyl moieties (Table 3). Significant quantities of these unknowns were also observed when 1 pM [5-3HlNNAL or WHINNK was incubated with rat liver microsomes, NADP+, and the NADPH regenerating system for 60 min a t 37 "C (Figure 2C,D). Subsequently, it was shown that unknown B, formed from NNK under these conditions, was the same as NNAL unknown A. The radiograms obtained for these mixtures were more complex than those obtained with pancreatic microsomes since rat liver microsomes are capable of catalyzing both the a-carbon hydroxylation and N-oxidation of these two nitrosamines (10,17). In the case of NNK, the majority of radioactive peaks were identified by coelution with unlabeled standards of keto acid, NNAL-N-oxide, diol, NNK-N-oxide, keto alcohol, and NNAL (Figure 2D). These results are comparable to what has been previously reported (10,17). However, previous studies have not indicated the presence of the unknown NNK metabolites we observed. We found that the separation of unknown A and B from keto acid using the pH 7 sodium phosphate mobile phase

NADP(H) Analogs of NNK and NNAL i n Microsomes

E

Q U L,

0)

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 603

c

L

c

0

l b ' 2b ' 3b

' 4b

I

sb

S'O

'0

I

. . . . . . . . . . . .

10

20

30

40

50

60

minutes Figure 2. Representative HPLC radiograms of [5-3HlNNAL(A and C) and [5-3HlNNK(Band D)metabolites formed in rat pancreatic (A and B) and liver microsomal incubations (C and D). The in vitro mixtures consisted of 1 p M [5-3HlNNAL and [ 5 - 3 H l q microsomal protein (1 mg/mL), N A D P , and an NADPH regenerating system. Incubations were carried out at 37 "C for 60 min. HPLC analysis was performed using HPLC method B.

minuter

Figure 3. Representative HPLC radiograms of [UHINNK (A and B)and [5-3HlNNAL (C) metabolites formed in rat pancreatic

(A) and liver microsomes (B and C). The in vitro incubation microsomixtures consisted of 1pM Kb3H1NNK or [C~-~H]NNAL, mal protein (1mg/mL), and 5 mM NADP+. Incubations were performed at 37 "C for 60 min. HPLC analysis was performed using HPLC method B (A and B) and method C (C). Table 3. Levels of NNK and NNAL Unknown Metabolites Formed from Methyl and Pyridyl Tritiated NNK and NNAL in Pancreatic Microsomal Incubation@ DmoVmL NNK NNK NNK unknowns substrate unknown A unknown B unknown C B and C [5-3H]NNAL 13.1 [Me3H]NNAL 11.8 [EI-~H]NNK 12.4 7.7 20.1 [Me-3HlNNK 8.7 8.7 17.4

"AL

=L3H1NNK or E3H1NNAL (1 pM) was incubated with rat pancreatic microsomes (1mg/mL), 5 mM NADP,1unit of glucose6-phosphate dehydrogenase, and 25 mM glucose 6-phosphate in 100 mM potassium phosphate buffer (pH 7.4) containing 3 mM MgCl2 and 1mM EDTA. Incubations were conducted for 60 min a t 37 "C.

depended on column conditions. Newer columns were more likely to provide base-line separation. These two derivatives were further separated by reanalyzing the

mixture at pH 6, with the unknown now eluting a t 22 min and keto acid at 19 min. It was difficult to determine if NNK unknown C was formed in rat liver microsomal incubations. Unknown C elutes in the vicinity of NNAL-N-oxide, the relative retention time depending on column conditions. HPLC analysis at pH 6 caused unknown C to elute approximately 5 min later with no effect on the retention time of NNAL-N-oxide. However, under these conditions, unknown C coelutes with diol. A number of radioactive peaks were observed when [5-3HlNNAL was incubated with rat liver microsomes and the complete NADPH regenerating system (Figure 2 0 . The earliest eluting peak had a retention time comparable to unknown A. The formation of NNAL-Noxide and diol was confirmed by coelution of radioactive peaks with the corresponding unlabeled standards (HPLC methods B and D). There was also a radioactive metabolite that eluted with the keto alcohol and lactol standards. This NNAL metabolite disappeared upon inclusion of bisulfite in the pH 6 buffer of HPLC method D. Since bisulfite reacts with aldehydes, the retention time of keto alcohol is changed by the presence of bisulfite. However, presumably the hydroxy aldehyde that is in equilibrium with lactol (see Figure 1) can react with bisulfite, causing a shift in retention time. The resulting adduct is unstable, so no distinct new radioactive peak is observed.2 The identity of NNAL-N-oxide, diol, and lactol were confirmed by LC-electrospray mass spectral analysis and HPLC with diode-array detection. A solution containing the ethyl acetate-soluble NNAL metabolites had components with retention times similar to those of the NNAL-N-oxide, diol, and keto alcohol (lactol) standards. Molecular ions (M H+)obtained for these compounds

+

S. E. Murphy, personal communication.

Peterson et al.

604 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 508

A

loo[ 75

25

/

100 200 300 400 500 600 700 604

I

lo@ 5

OO

1.0 2.0 Concentration (mM)

3.0

Figure 4. Concentration dependence of NNAL and NNK metabolite formation. The NNAL incubationscontained [5-3HlNNAL (0-2000 pM), 5 mM NADP+, an NADPH regenerating system, and rat liver microsomes (1 mg of proteidml). Incubations were conducted at 37 "C for 60 min. ("&)ADP+, 0; NNAL-N-oxide,.; diol, 0 ; and lactol, A. The NNK study was conducted with [5-3H]NNK(0-3000pM), 5 mM NADP+,and rat liver microsomes (1 mg of proteidml). Incubations were conducted at 37 "C for 20 min. (NNK)ADP+,A. matched those of standards: mlz 226, mlz 168, and mlz 166, respectively. W spectra of the unknowns were matched to standards for the predicted NNAL metabolites: NNAL-N-oxide, A,,,= 211 and 255 nm; diol, Amax 200 and 260 nm; and lactol, A,,,= 210,235, and 260 nm. Methylene hydroxylation of NNAL (lactol formation) was greater than methyl hydroxylation (diol) a t the 1pM concentration (Figure 4). This contrasts to what has been reported for NNK in rat liver microsomes; methyl hydroxylation (keto alcohol) is slightly greater than methylene hydroxylation (keto aldehyde) (I7). The HPLC radiograms obtained for [Eb3H]NNK and [5-3HlNNALwere significantly simplified upon exclusion of the regenerating system from the rat liver microsomal incubations (Figure 3). The radiograms from the NNK incubations contained only unknown B and NNAL (Figure 3B). Unknown A was the only radioactive peak formed from NNAL (Figure 3 0 . Unknowns A and B were isolated by preparative HPLC of large-scale incubations of [5-3HlNNALor [5-3HlNNK with liver microsomes and NADP+. Structural analysis of these unknowns indicated that they were analogs of NADP+ in which the nicotinamide portion had been replaced with NNAL (unknown A) or NNK (unknown B) [(NNAL)ADP+and (NNK)ADP+, respectively]. The NMR spectra of the unknowns in DzO contained all the signals associated with NNAL or NNK (Table 1). However, the pyridyl protons were shifted downfield relative to the parent compounds. In addition, there were two aromatic singlets at approximately 8.4 and 8.1 ppm, corresponding to the 8- and 2-protons of adenine. The spectra also displayed signals for the two ribosyl sugar residues. Electrospray ionization mass spectral analysis of the metabolites yielded molecular ions at mlz 831 for un-

75

t

I / 4 0490

0

;

'

1

','II

829

I

,L

NADP(H) Analogs of NNK and NNAL in Microsomes

410

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 605

L!:cH3 L::cH3

I

I

I

200

3iO

4iO

500

Wavelength (nm)

Figure 8. W spectra of NADPH (-) and (NNKIADPH (unknown C; - - -) from pancreatic microsomal incubations of NNK, NADP+, and an NADPH regenerating system in 20 mM sodium phosphate buffer (pH 7).

HO-P=O

I

OH

Figure 6. Fragmentation patterns for NADP+, (NNAL)ADP+, and (NNK)ADP+.

200

300

400

Wavelength (nm)

Figure 7. UV spectra of NADP+ (-), (NNK)ADP+ (- - -), and (- -1 in 10 mM ammonium acetate solution. ("&)ADP+ (NNK)ADP+ and (NNAL)ADP+ were isolated from rat liver

microsomal incubations of NADP+ with NNK and NNAL, respectively.

Table 4. Effect of Basic, Acid, and Neutral Thermal Hydrolysis on ("&)ADP+ and (NNK)ADP+Formed in Rat Liver Microsomal Incubations compound (NNKUDP+

hydrolysis conditions"

neutral 0.1 N HC1 0.1 N NaOH (NNAL)ADP+ neutral 0.1 N HCl 0.1 N NaOH 20-22-mm fractionC neutral 0.1 N HCl 0.1 N NaOH

*

NADP NNAL NNK analog (dpm) (dpm) (dpm) 5890 4639

1639 884 5084

981 3516 3819 3645 3819 3629

a C o m ~ o u n dwere ~ heated at 80 "C for 0 min in O m M phosphate buffer (pH 6), 0.1 N HC1, or 0.1 N NaOH. (NNK)ADP+ and ("&)ADP+ were isolated from incubations of 1 pM [EI-~HINNKor [5-3HlNNAL with rat liver microsomes and 5 mM NADP+ using HPLC method F. 20-22-min fraction isolated from incubations of L3H]NNKwith rat liver microsomes, 5 mM NADP+, and an NADPH regenerating system using HPLC method F. This fraction was thought to contain (NNK)ADP+.

ing system. Comparable UV spectra have been reported for other NAD analogs (30, 31). Consistent with the assigned structure, fractions containing the metabolites formed from 1pM [5-3H]NNKor [5-3H]NNAL in the presence of rat liver microsomes released [5-3H]NNKand [5-3H]NNAL,respectively, when heated in 0.1 N NaOH for 30 min (Table 4) (32). Lesser amounts of the parent nitrosamines were released under neutral thermal or 0.1 N HCI hydrolysis. Comparable results were obtained for unknowns A and B isolated

from rat pancreatic microsomal incubations of NNAL or NNK and unknown A isolated from rat liver microsomal incubations (data not shown). These incubations were performed in the presence of NADP+ and an NADPH regenerating system. However, NNAL was released upon basic hydrolysis of NNK "unknown B isolated from rat liver microsomal incubations of [5-3H]NNK that included NADP+ and the regenerating system. This result demonstrates that "unknown B" observed in the rat liver microsomal incubations is not (NNK)ADP+but (NNAL)ADP+. This surprising observation is consistent with the rapid reduction of NNK to NNAL and the relatively slow formation of the NADP+ analog in rat liver microsomes (data not shown). Unknown C is believed to be the corresponding NADPH analog, (NNKIADPH. The evidence for this assignment is as follows. Incubations of (NNK)ADP+ with glucose-6-phosphate dehydrogenase and glucose 6-phosphate generate a new species that has the same retention time as unknown C. The UV spectrum of this metabolite formed in pancreatic microsomal incubations was similar to that of NADPH (Figure 8). The major difference is that the spectrum contains a a,, at 365 nm instead of 340 nm. These results compare favorably to those reported for (3-acety1pyridine)ADH. In this case, the UV spectrum of the reduced compound also has a Am= at 365 nm (32). A partial 'H NMR spectrum of unknown C was obtained; the aromatic signals of this derivative (singlets at 8.6, 8.2, and 7.6 ppm) lacked the pyridinium protons observed in the spectrum of (NNK)ADP+. The aromatic signals observed were similar to those observed for NADPH (singlets at 8.75, 8.2, and 7.3 ppm). The concentration-dependent formation of (NNAL)ADP+ and (NNK)ADP+ was determined in rat liver microsomes. The NNAL studies were performed in the presence of the NADPH regenerating system. The NNK studies were conducted in the absence of the regenerating system since we wanted to look at the formation of (NNK)ADP+,not (NNAL)ADP+,the observed product in rat liver microsomal incubations containing the regenerating system. As shown in Figure 4, the formation of these analogs is linear with NNAL or NNK concentration. Similar analogs have been reported for other pyridinecontaining compounds. Rabbit liver microsomes catalyzed the formation of the nicotine NADP+ derivative (30). The formation of this derivative was linear with nicotine concentrations up to 50 mM. Other chemicals that have been shown t o participate in this reaction include cotinine (33),3-acetylpyridine (32),nicotinic acid (34), and isonicotinic acid hydrazide (35). These ex-

Peterson et al.

606 Chem. Res. Toxicol., Vol. 7, No. 5, 1994

(NNK)ADP+

-

Enz. ribose ADP

I

Hzo

HO .ribose. ADP

ribace-AOP

ribosc-ADP (NNKIAOPH

ribose-ADP 3

I

I

ribose-ADP

ribose-ADP

(NNK)AOP*

(NNAL)AOP*

I

O

o=p+QHO

OH

T

I

0

5

15

25

35

45

minutes

Figure 9. Representative HPLC radiograms of incubation mixtures of 1,uM [5-3H]NNK(A) or [5-3H]NNAL(B)with 5 mM NADP+and porcine brain NAD glycohydrolase. Mixtures were analyzed using HPLC method C.

change reactions have also been reported in vivo for compounds such as cotinine (361, 3-acetylpyridine (371, and 6-aminonicotinamide (38). The enzyme NAD glycohydrolase catalyzes these transglycosylation reactions (32-35). In order to determine if NNK and NNAL are substrates for this enzyme, the radiolabeled nitrosamines were incubated with porcine brain NAD glycohydrolase and NADP+. Each radiogram contained a single radioactive product (Figure 9) that coeluted with microsomally derived (NNK)ADP+ and ("&)ADP+. Product formation was dependent on the presence of NADP+ and enzyme. These products released the parent nitrosamine with basic but not neutral or acid hydrolysis (data not shown). These results are consistent with the formation of the transglycosylation products, NNK(ADP)+ and "&(ADP)+. NAD' is also a substrate for NAD glycohydrolase (39, 40). Incubations of rat liver microsomes with NNK in the presence of 5 mM NAD+ generated a radioactive metabolite that eluted 7 min after (NNK)ADP+ (HPLC method C ) . However, the levels of this product were 5 times less than observed with NADP+ (2.0 and 10.1 pmol/ mL, respectively). Similar results were observed with glycohydrolase (2.1 and 7.5 pmol/mL, respectively). Our data demonstrate that rat liver and pancreatic microsomes catalyze transglycosylation reactions, resulting in the formation of (NNK)ADP+ and ("&)ADP+. The involvement of microsomal NAD glycohydrolase in this reaction is supported by the ability of purified porcine brain NAD glycohydrolase to mediate the same reactions. NAD glycohydrolases are membrane-bound enzymes that catalyze the cleavage of the pyridine-ribose bond of NAD(P)+ (40-42). Mechanistic studies support the formation of an enzyme-stabilized ADP-ribosyl oxonium ion intermediate (43, 44). This intermediate can react either with HzO to produce ADPR or with nicotinamide or compounds such as NNK or NNAL, to generate NADP+ or an analog (Figure 10). (NNK)ADPH most likely comes from the reduction of (NNK)ADP+ since NADPH is not a substrate for NAD glycohydrolase (45). The physiological role of these enzymes is not clear. Their involvement in NAD(P)+ catabolism and calcium

H

HO OPO:

Figure 10. Proposed mechanism of NADP+ analog formation from NNK or NNAL as catalyzed by NAD glycohydrolase.

homeostasis has been proposed ( 3 9 , 4 6 , 4 7 ) .The potential role of these NADP analogs in the toxicity and carcinogenicity of NNK and NNAL is unknown. 3-Acetylpyridine is toxic to mice (37). The toxicity can be reversed upon coadministration of nicotinamide to the animals. The toxicity is also accompanied by alterations in NAD levels and NAD-related enzyme activity. 6-Aminonicotinamide is also able to interfere with NADrequiring enzyme activity in vivo, presumably as a result of the formation of the corresponding NAD and NADP analogs (38). Formation of the NAD derivative of W methylnicotinamide precedes the induction of human promyleocytic leukemia HL-60 cell maturation caused by this compound (48). NAD glycohydrolases belong to a class of enzymes that catalyze the cleavage of the pyridine-ribose bond of NAD(P)+ and the transfer of the ADP-ribosyl group to an acceptor. The acceptor in the case of NAD glycohydrolases is HzO or a nitrogen-containing compound such as nicotinamide. Mono(ADP-riboseltransferasescatalyze the transfer of a single ADP-ribose molecule. Poly(ADPrib0se)polymerases catalyze the synthesis of ADP-ribose polymers. Proteins are the acceptor molecules for both of these enzyme families (49). Inhibitors of poly(ADPribose)polymerase can affect many cellular processes such as differentiation (50,511,DNA replication (52),malignant transformation (53,541,recovery from DNA damage (55,561,and sister chromatid exchange (57). Poly(ADPribose)polymerase also has NAD glycohydrolase activity (58). Inhibitors of this enzyme enhanced diethylnitrosamine tumorigenesis in rats (59). It would be interesting if NNK, NNAL, or the corresponding N A D P analogs are able to inhibit the activity of these enzymes. The inability of pancreatic microsomes to support a-hydroxylation reactions of NNAL and NNK is consistent with previous reports for other nitrosamines (60). Recently, Kokkinakis and co-workers reported that cultured pancreatic duct cells are capable of activating N-nitrosobis(2-oxopropy1)amine (BOP) and N-nitroso(2hydroxypropyl)(2-oxopropyl)amine (HPOP) to DNA-reactive species via a-hydroxylation to a limited extent (61). The low activity of a-hydroxylation of NNK and NNAL in pancreatic microsomes suggests that this tissue is not

NADP(H) Analogs of NNK and NNAL in Microsomes capable of activating these nitrosamines. If adducts are detected in pancreatic DNA of animals treated with NNK or NNAL, it is possible that activated metabolites of "K and NNAL are transported to the pancreas via the blood stream as has been postulated for BOP and HPOP (62). We are currently exploring this possibility.

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 607

(14) Peterson, L. A., Mathew, R., and Hecht, S.S.(1991) Quantitation of microsomal a-hydroxylation of the tobacco-specificnitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone. Cancer Res. 51, 5495-5500. (15) Smith, T. J., Guo, Z., Hong, J.-Y., Ning, S. M., Thomas, P. E., and Yang, C. S.(1992) Kinetics and enzyme involvement in the metabolism of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK)in microsomes of rat lung and nasal mucosa. Carcinogenesis 13, 1409-1414. Acknowledgment. We would like to thank the (16) Hong, J.-Y., Ding, X., Smith, T. J., Coon, M. J., and Yang, C. S. following people for their contributions to these studies: (1992) Metabolism of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK),a tobacco-specific carcinogen, by rabbit nasal Ketaki Patel for technical assistance; Dr. Fred Kadlubar microsomes and cytochrome P450s NMa and NMb. Carcinogenat the National Center for Toxicological Research in Little esis 13, 2141-2144. Rock, AR,for helpful discussions; Dr. Dhimant Desai for (17) Guo, Z., Smith, T. J., Thomas, P. E., and Yang, C. S. (1992) preparation of unlabeled NNK and NNAL; and the Metabolism of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone by Research Animal Facility at AHF for isolation of tissues. inducible and constitutive cytochrome P-450 enzymes in rats. Arch. Biochem. Biophys. 298,279-286. This study was supported by Grant CA-44377 from the (18) Hecht, S. S., and Trushin, N. (1988) DNA and hemoglobin National Cancer Institute. The Research Animal Facility alkylation by 4-(methylnitrosamino)-l-(3-pyridyl)-l-bu~one (NNK) is partially supported by National Cancer Institute and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanol(NNAL) in Cancer Center Support Grant CA-17613. F344 rats. Carcinogenesis 9, 1665-1668. (19) Hecht, S. S., Jordan, K. G., Choi, C.-I., and Trushin, N. 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