Disposition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in bile duct-cannulated rats: Stereoselective metabolism and tissue distribution
Zheng Wu1,
Pramod Upadhyaya2,
Steven G. Carmella2,
Stephen S. Hecht2 and
Cheryl L. Zimmerman1,3
1 College of Pharmacy and
2 Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
 |
Abstract
|
---|
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a chiral compound, and the primary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major carcinogen in tobacco smoke. The goal of the present work was to study the pharmacokinetics and stereoselective metabolism and tissue retention of NNK and NNAL in the rat. Groups of rats were dosed with [5-3H]NNK (n = 3) or racemic [5-3H]NNAL (n = 3) at a target dose of 8.45 µmol/kg and were killed at selected time points for tissue collection. Separate groups of rats (n =5 per group) received the same dose of either NNK or NNAL and serial sampling of blood, bile and urine was carried out over 24 h. All samples were analyzed by C18 reversed-phase HPLC with gradient elution and radioflow detection. A gas chromatographthermal energy analyzer (GCTEA) was used to separate the (R)-/(S)-NNAL enantiomers. Racemic NNAL and NNK had large volumes of distribution (321 ± 137 ml for NNK and 2772 ± 1423 ml for NNAL) and similar total body clearances (12.8 ± 2.0 ml/min for NNK and 8.6 ± 2.6 ml/min for NNAL). The results indicated that the enantiomers of NNAL are stereoselectively metabolized and excreted. The glucuronide of (R)-NNAL, ((R)-NNAL-Gluc) was identified as the major metabolite in the bile after administration of either NNK or NNAL. (R)-NNAL was the major NNAL enantiomer in the bile or urine samples. At 24 h after racemic NNAL administration, NNAL comprised an average of 75.4% of total radioactivity in the lung with an (S)-/(R)-ratio of >20. The stereoselective localization of (S)-NNAL to lung tissue may contribute to the lung selectivity of NNK carcinogenesis. The present studies suggest a need to look beyond metabolic activation as the sole mechanism for lung carcinogenesis.
Abbreviations: GCTEA, gas chromatographthermal energy analyzer; hydroxy acid, 4-hydroxy-4-(3-pyridyl)butyric acid; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL-Gluc, [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-ß-O-D-glucosiduronic acid; NNAL-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; keto acid, 4-oxo-4-(3-pyridyl)butyric acid; TMS, bis-trimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane
 |
Introduction
|
---|
Lung and bronchial cancer was expected to cause 157 000 deaths in the year 2000 in the United States (1). Tobacco-alkaloid-derived nitrosamines, called `tobacco-specific nitrosamines', are present in substantial quantities in tobacco smoke. Among the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its primary metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N'-nitrosonornicotine are the most carcinogenic (2,3).
NNK is a potent and selective inducer of adenocarcinoma of the lung in rodents (3). In F344 rats, lung tumors were induced regardless of the route of administration, including in drinking water, or by subcutaneous injection, gavage, oral swabbing or intravesicular injection (3). The lowest total dose of NNK for induction of lung tumors was around 1.8 mg/kg (8.7 µmol/kg) (4).
NNK metabolism has been studied extensively (3,510). Carbonyl reduction, pyridine oxidation, and
-hydroxylation are the three major routes of metabolism (Figure 1
). Quantitatively, carbonyl reduction to NNAL is the most active pathway in vivo (3). NNAL can be subsequently conjugated to form NNAL-glucuronide, which is excreted in the urine. Formation of NNK-N-oxide and NNAL-N-oxide are the results of pyridine oxidation. Both metabolites can be detected in the urine of humans and rodents (11), but quantitatively, this pathway is of less significance than carbonyl reduction. The keto acid and hydroxy acid are the end products of the
-hydroxylation pathways of NNK and NNAL, respectively (12,13); this is the route by which DNA-binding intermediates are formed (3,14). The keto acid and hydroxy acid metabolites are largely excreted in the urine in rats, mice and patas monkeys (12,13).
NNAL is an enantiomeric compound (15) while NNK is prochiral. In addition, NNK metabolism to NNAL is reversible (16). The consequence is that the interconversion of NNK and NNAL is a four-fold kinetic process: NNK to (S)-NNAL, NNK to (R)-NNAL, (S)-NNAL to NNK, (R)-NNAL to NNK. The in vivo pharmacokinetic behaviors of NNK, (S)-NNAL and (R)-NNAL will be determined by the relative rates of these conversions and it is possible that the rate of metabolism of each of the four processes will be different. The situation becomes more complicated as the conversion and reconversion of each compound also competes with parallel pathways of metabolism such as
-hydroxylation, glucuronidation and N-oxidation.
Recent studies indicate that stereochemical aspects of NNAL and NNAL-Gluc formation could play an important role in the metabolic detoxification of NNAL as well as its carcinogenic activity (15,17). Bioassay results in A/J mice indicate that the carcinogenic potency of (S)-NNAL was equal to that of the parent compound NNK, while (R)-NNAL was less potent (17). (S)-NNAL is the predominant form found in rat and human microsomal incubations with NNK (15,18). It was hypothesized that the carcinogenic difference of (S)- vs. (R)-NNAL was caused by the difference of the in vivo disposition profile (a pharmacokinetic issue) of the two enantiomers rather than a difference in their intrinsic activity (a pharmacodynamic issue).
The specific objectives of the present study were to: (i) Characterize the general pharmacokinetics of NNK and racemic NNAL after their separate intravenous administration to chronically bile duct-cannulated rats; (ii) Investigate the balance between biliary and urinary excretion of NNK, NNAL and their metabolites; (iii) Determine the extent of stereoselective metabolism and disposition of NNAL and its metabolites.
 |
Materials and methods
|
---|
Chemicals
Unlabeled NNK and [5-3H]NNK (1.8 Ci/mmol) were purchased from Chemsyn Science Laboratories (Lenexa, KS). ß-Glucuronidase, type IXA, and pentobarbital were purchased from Sigma Chemical Co. (St Louis, MO). Racemic [5-3H]NNAL and unlabeled NNAL were synthesized as previously reported (19). Picofluor-40 was purchased from Packard Instruments (Meridan, CT). Bis-trimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane (TMS) was purchased from Pierce (Rockford, IL). All other chemicals were of analytical grade.
Animals
Male Fisher 344 rats (249 ± 26 g, n = 22) were obtained from Harlan (Indianapolis, IN). They were housed two per cage under standard conditions (20 ± 2°C, 12 h lightdark cycle), and were given food and water ad libitum.
Animal treatment
Rats were anesthetized with pentobarbital (50 mg/kg, i.p.). The body temperature was monitored with a rectal probe and kept constant by means of a heating pad. For rats in the first group, the right femoral artery and vein were cannulated with heparinized polyethylene tubing (PE-50, Clay Adams, Parsippany, NJ); the bile duct was also cannulated with polyethylene tubing (PE-10, Clay Adams). The tubing was then passed through a subcutaneous tunnel to the nape of the neck and tied to a tether (Model 56-1456, Harvard Apparatus, South Natick, MA). Each rat was placed in a metabolic cage (Model 52-6707, Harvard Apparatus) and the arterial tubing was connected to an infusion pump (Model 975, Harvard Apparatus) with which saline was infused at 1 ml/h. The rats were allowed to recover overnight (at least 12 h) before the experiment. For rats in the second and third group, only the bile duct and the femoral vein were cannulated.
The dosing solution for each rat was prepared in 0.9% sodium chloride by diluting
100 µCi of [5-3H]NNK or [5-3H]NNAL with the respective unlabeled compound to give a total dose of
8.45 µmol/kg. The actual dose was 8.44 ± 1.5 µmol/kg (n = 11) for NNK and 8.48 ± 0.56 µmol/kg (n = 11) for racemic NNAL. The dose was given through the femoral vein as a bolus at a rate of 2 ml/min. Each rat received
200 µl of dosing solution.
For each compound, three groups of rats were dosed separately. In the first group, five animals were used for blood, urine and bile sampling. The rats were sampled serially over 24 h. At the end of the 24 h sampling period, the rats were killed and the lung, liver and kidney tissues were collected. In the second and third group, three animals each were dosed and subsequently killed at 1 or 4 h after dosing. The lung, liver and kidney tissues were collected.
Blood samples (200 µl) were collected through the arterial cannula into microcentrifuge tubes at 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360 min post-dose for the NNK study and at 5, 10, 15, 30, 45, 60, 90, 120, 180, 360 min post-dose for the NNAL study. The tubes were immediately centrifuged at 10 000 g for 5 min and the resultant plasma samples were put on ice. Urine was collected at intervals of 02, 24, 48, 812, 1224 h post-dose for the NNK study and at intervals of 02, 24, 46, 68, 812, 1224 h after administration for the NNAL study. Bile was collected at intervals of 00.5, 0.51, 12, 23, 34, 46, 68, 812, 1224 h post-dose for both studies. A 10 µl aliquot of each sample was removed for liquid scintillation counting (LSC), while the rest was stored at 20°C until analysis. After completion of the study, the rats were killed by an overdose of pentobarbital, and lung, liver and kidney tissues were obtained.
Tissue extraction of NNAL and metabolites
Lung, liver and kidney tissues were rinsed in 0.9% NaCl solution, blotted dry and weighed. The tissues (approximately 1 g for the lung, 2 g each for the liver and kidney) were minced with scissors and homogenized in 8 ml of ice-cold 0.1 N HCl with a PowergenTM 125 tissue homogenizer (Fisher Scientific) at the highest speed for 2 min. The homogenate was centrifuged at 5000 g for 15 min. A 6 ml aliquot of supernatant was neutralized with 1 N NaOH. The resulting solution was treated with 6 ml of cold methanol and centrifuged at 5000 g for 15 min. A 10 ml aliquot of supernatant was dried on a SupelcoTM (Bellefonte, PA) vacuum manifold; the residue was subsequently reconstituted to 300 µl with 0.9% NaCl and stored at 70°C until the day of analysis (13).
Liquid scintillation counting
Samples of bile, urine and plasma were aliquoted (10 µl) and added to 5 ml Picofluor-40 (Packard Instruments) liquid scintillation fluid. Aliquots of tissue homogenate (100 µl) were first solubilized with 1 ml TS-1 tissue solubilizer (Research Products International, Mount Prospect, IL). The solubilization took place on a block heater at 80°C for 60 min. A 100 µl aliquot of the solubilized tissue was then added to 5 ml Picofluor-40. Liquid scintillation counting was conducted with a Model 3801 liquid scintillation counter (Beckman Corp., Fullerton, CA) with an average counting efficiency of 40%.
Analysis of NNK and metabolites
Plasma samples were prepared for HPLC by filtration through Amicon Centrifree Micropartition Filters (Amicon, Beverly, MA). Typical urine and bile samples were spiked with non-radiolabeled standard and injected directly on HPLC. The volume of injection for plasma samples was typically 100 µl (containing
10 000 d.p.m.); the volumes of injection for bile and urine samples were typically 50100 µl (containing
100 000 d.p.m.). For tissue samples the injection volume was usually 100 µl (containing
10 000 d.p.m.). NNK and its metabolites were separated by reversed-phase HPLC with a Millipore-Waters Associates HPLC system (Milford, MA), equipped with a 4.6 x 250 mm PartisphereTM 5 µm C18 column (Whatman, Clifton, NJ) and a Model 490 (Waters) UV/visible detector operated at 254 nm. Radioflow analysis was carried out in tandem with the use of a ß-RAM radioactivity flow detector (IN/US systems, Fairfield, NJ). Separation was accomplished with a 20 mM sodium phosphate buffer (pH = 7.0)/methanol gradient increasing from 07% methanol over 15 min, a 15 min hold, then a gradient increasing from 735% methanol over 3075 min. The flow rate was 1 ml/min. NNK and metabolites were identified by coelution with known unlabeled standards (13). Glucuronide conjugates were confirmed by treatment of a portion of each bile or urine sample (typically 100 µl) with 500 units of ß-glucuronidase at room temperature for 24 h with subsequent analysis by HPLC (13). Picofluor-40 (Packard Instruments) liquid scintillation cocktail was used in the radioflow detector and was pumped at flow rate of 4 ml/min.
The calculation of the concentration of each compound in the sample of interest was done by a three-step process. The first step involved counting the total radioactivity by means of LSC, with the total radioactivity representing parent and metabolite concentrations in a specific sample. In the second step, the parent and metabolites were separated by HPLC and the peak areas of the individual compounds were determined by radioflow detection. The integrated area under each peak was divided by the total integrated area of the HPLC run. In the third step, the results from the first two steps were multiplied together to obtain the total counts for each compound in the sample of interest. This was then converted to concentration of each compound with the use of the original specific activity.
Analysis of (R)-NNAL AND (S)-NNAL by GCTEA
NNAL from various samples was further purified by means of HPLC collection. These collections were performed with a single PhenomenexTM (Torrance, CA) 3.9 x 300 mm C18 analytical column with a Gilson (Middleton, WI) fraction collector. The elution solvent was a mixture of ammonium acetate buffer (10 mM, pH 6.6) and methanol with a flow rate of 1 ml/min. The elution program consisted of a gradient of 07% methanol over 7 min, then held at 7% methanol for 15 min, then from 22 to 50 min, the gradient increased from 7 to 35% methanol. NNAL eluted at 4143 min. After collection, the eluant was dried on a SupelcoTM vacuum manifold; it was subsequently reconstituted in 100 µl methanol and stored at 70°C until the day of analysis.
On the day of analysis,
50 µl of the reconstituted solution was transferred to a 100 µl GC specific glass-lined microvial (#225195, Wheaton, Millville, NJ), and concentrated to dryness on a model SVC200H Speedvac concentrator (Savant Instruments, Farmingdale, NY) at room temperature. To the residue was added 10 µl TMS. The mixture was mixed thoroughly and heated to 50°C for 1 h, then 2 µl was injected directly on the GC.
The separation of (R)- and (S)-NNAL was accomplished by means of GC utilizing a Hewlett-Packard (Palo Alto, CA) Model 5890 instrument interfaced with a Model 610 Thermal Energy Analyzer (TEA) (Thermedics Inco, Woburn, MA) and a model D-7500 integrator (Hitachi Instruments, Danbury, CT). The TEA is a nitrosamine-selective detector. The GC was equipped with a CyclosiläTM column (30 m x 0.25 mm, J and W, Folsom, CA), with a 0.32 mm x 1 m retention gap. The carrier gas was He (head pressure, 12 psi) and the oven temperature was programmed as follows: 60°C for 2 min, then the temperature was increased at a rate of 12°C/min to 166°C which was held until 90 min. The injection port temperature was 225°C. The injection was done by pulse splitless injection. The pulse pressure was 50 psi, pulse time was 0.5 min, purge time was 0.75 min and purge flow was 60 ml/min. Chromatograms of the separation of (R)- and (S)-NNAL have been previously published (20), but it should be noted that the assignments were reversed in that publication.
Pharmacokinetic analysis
The plasma concentration vs. time data for NNK and NNAL were analyzed by non-compartmental analysis (21). The slope of the terminal phase of the urinary excretion rate vs. time curve was determined by fitting the log urinary excretion rate vs. time data to a mono-exponential function with least squares linear regression (Microsoft Excel, PC version 97, Microsoft, Redmond, WA). The terminal rate constant,
, was determined from the slope. The area under the plasma concentrationtime curve from time zero to time t (AUC (0t)) was determined by the linear trapezoidal rule up to the last measured concentration. The AUC (t
) was determined by dividing the last measured concentration by the terminal urinary rate constant
. The AUC (0
) was the sum of the two partial AUCs. The urinary elimination half-life (t1/2) was calculated as 0.693 divided by
. The total body clearance (CL) was calculated as:
Steady state volume of distribution was calculated as:
where AUMC is the area under the first moment of the plasma concentrationtime curve. Renal clearance (CLr) was calculated as:
where Xu (0t) was the amount of drug collected in the urine during the collection period up to time t. Biliary clearance (CLb) was calculated as:
where Xb (0t) was the amount of drug collected in the bile during the collection period up to time t.
Statistical analysis consisted of analysis of variance (ANOVA) or paired Student's t-test which was conducted with Sigma Stat (Version 3.06, Jendal Scientific, San Rafael, CA).
 |
Results
|
---|
The pharmacokinetics of NNK and racemic NNAL were studied in chronically bile duct-cannulated rats. After surgery, the rats were allowed to rest overnight while the bile flow was monitored. If an intermittent or slow bile flow was noticed, the rat was excluded from the study. The bile flow was constant during the experiment, at
0.7 ml/h (data not shown).
After 24 h, roughly 85% of a total dose of NNK (17.5 ± 3.8% from bile and 67.6 ± 10.1% from urine) was recovered, while
60% of the total dose (19.5 ± 2.6% from bile and 42.6 ± 8.2% from urine) was recovered after NNAL administration. The plasma concentration time-profile (not shown) indicated that NNK apparently followed a monoexponential decline after NNK administration. Pharmacokinetic analysis (Table I
) indicated that NNK had a short urinary half-life, a large volume of distribution (321 ± 137 ml) and a total body clearance of 12.8 ± 2.0 ml/min. NNAL had a total body clearance of 8.65 ± 2.6 ml/min, and a very large volume of distribution (2772 ± 1423 ml). Unchanged NNK as well as racemic NNAL had minimal biliary clearance. Unchanged NNK was essentially not cleared in the urine while NNAL had a renal clearance equivalent to roughly 17% of its total body clearance.
Since the compounds could be quantitated in the urine for up to 24 h, the urinary excretion rate vs. time profiles were utilized to calculate the apparent elimination half-lives of the administered compounds (Table II
). The apparent elimination half-life of NNK following NNK administration was the shortest,
2 h. The keto acid and NNAL, NNK's major urinary metabolites, as well as hydroxy acid and NNAL-N-oxide (NNAL's metabolites) each had an apparent elimination half-life statistically similar to that of NNK, indicating a formation rate-limited process. The elimination process for NNK-N-oxide and NNAL-N-oxide appeared to be rate-limiting in their pharmacokinetics as indicated by the longer half-lives over NNK. However, this difference did not reach statistical significance.
The extent of biliary excretion of NNK and its metabolites is shown in Figure 2a
. (R)-NNAL-Gluc was the major metabolite in the bile, consisting of 13.8 ± 3.4% of the total NNK dose and 15.7 ± 3.4% of the total racemic NNAL dose. Nearly all NNAL-Gluc was excreted as the (R)-diastereomer. The identity of the glucuronide metabolites were confirmed by treating the sample with ß-glucuronidase and analyzing the amount of NNAL released. NNK, (R)-/(S)-NNAL, hydroxy acid, keto acid and NNAL-N-oxide were also detected in the bile in small quantities. In summary, the biliary excretion profiles after either NNK or NNAL were very similar. (R)-NNAL-Gluc comprised roughly 85% of the total metabolites excreted in the bile.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2. (a) Biliary metabolite excretion after administration of NNK (closed bars) or racemic NNAL (open bars). (b) Urinary metabolite excretion after administration of NNK (closed bars) or racemic NNAL (open bars). Data are presented as mean ± SD, n = 5. HA, hydroxy acid; KA, keto acid; LOX, NNAL-N-oxide; R-GL, (R)-NNAL-Gluc; S-GL, (S)-NNAL-Gluc; KOX, NNK-N-oxide.
|
|
The urinary excretion profile (Figure 2b
) was markedly different from that of the bile. After NNK administration, NNK's
-hydroxylation product, keto acid, was the major metabolite excreted, consisting of 26 ± 3.3% of the total dose and 38.4 ± 4.9% of the metabolites excreted in the urine. The urinary excretion of NNAL and its
-hydroxylation product, hydroxy acid, made up
30% of the total dose after NNK administration. The urinary excretion profile following racemic NNAL administration was similar to that of NNK except that markedly less keto acid was excreted (9.2 ± 2.2% of the total dose). This indicates significant reversible metabolism from NNAL to NNK, as the keto acid comes mainly from NNK, and not from hydroxy acid (22). NNAL excretion after NNAL administration (12.9 ± 3.9% of total dose) was only slightly greater than after NNK administration (8.9 ± 2.8% of total dose). Only
1% of the dose of either compound was excreted in urine as NNK.
Because of the marked difference in the carcinogenic potential of the NNAL enantiomers, the NNAL (S)- vs. (R)- ratios in plasma, urine, and bile samples were evaluated (Figure 3
). Unquestionably, (R)-NNAL was the predominant form excreted. The (S)/(R) enantiomeric ratio for NNAL was below unity for all urine and bile samples regardless of the compound administered. NNAL could only be sampled in blood for a relatively short period of time due to its low concentration compared with that in the bile and urine. The (R)- and (S)- forms of NNAL were almost equal ((S)/(R) ratio 0.74 ± 0.18) at 5 min after NNK administration but the (R)-form was in greater quantities in plasma as early as 5 min after NNAL administration ((S)/(R) ratio 0.32 ± 0.08). The apparently decreasing trend in the plasma (S)/(R) enantiomeric ratios as a function of time after the administration of either compound did not reach statistical significance.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3. Enantiomeric ratio (S)/(R) of NNAL as a function of time in plasma (P), bile (B) and urine (U) samples following either NNK (closed bars) or racemic NNAL (open bars). Data presented as mean ± SD, n = 3.
|
|
The overall tissue retention of metabolites was evaluated (Table III
). On a per gram basis, lung apparently retained more dose 4 or 24 h following NNAL administration, although no statistical significance was reached. Tissue specific retention differences were not observed in samples following NNK administration.
View this table:
[in this window]
[in a new window]
|
Table III. Total amount of radioactivity-associated compounds (nmol/g, mean ± SD) remaining in selected tissues following NNK or racemic NNAL administration
|
|
The quantitation of metabolites in the tissue gave additional insight into the overall disposition of these compounds (Table III
, Figures 46

). In the liver samples collected 1 h after NNK dosing (Figure 4a
), hydroxy acid comprised 15.2 ± 6.2% of the total metabolites while the keto acid comprised 12.5 ± 5.8%. The hydroxy acid to keto acid ratio was approximately unity. Because hydroxy acid and keto acid are metabolites from NNAL and NNK metabolism, respectively, the ratio could serve as an indicator of the extent of the NNALNNK metabolism reversibility. The observed high hydroxy acid to keto acid ratio indicates extensive metabolism of NNK to NNAL following NNK administration. In the lung and kidney samples, keto acid recovery was higher (Figure 4a
), with a hydroxy to keto acid ratio of
0.5. These data indicate that the liver plays a greater role in the metabolic transformation of NNK to NNAL than kidney or lung, although NNK and NNAL were present in all three tissues. (R)-NNAL-Gluc was identified in the liver in relatively large quantities (11.9 ± 2.2% of total metabolites), as was suggested by its extensive biliary excretion.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4. (a) Each bar is the percent of total metabolites in a given tissue made up by a given metabolite 1 h after administration of NNK. (b) Each bar is the percent of total metabolites in a given tissue made up by a given metabolite 4 h after administration of NNK. Tissues include liver (closed bars), lung (hatched bars) and kidney (open bars). Data presented as mean ± SD, n = 3; #, mean, n = 2; +, n = 1. HA, hydroxy acid; KA, keto acid; LOX, NNAL-N-oxide; R-GL, (R)-NNAL-Gluc.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5. Enantiomeric ratio (S/R) of NNAL in liver (closed bars), lung (hatched bars) or kidney (open bars) as a function of time following either NNK or NNAL administration. Data presented as mean ± SD, n = 3; #, mean, n = 2. Note that the mean (n = 2) (S)/(R)-NNAL ratio in the lung 24 h after NNAL administration is 57.3.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6. (a) Each bar is the percent of total metabolites in a given tissue made up by a given metabolite 1 h after administration of NNAL. (b) Each bar is the percent of total metabolites in a given tissue made up by a given metabolite 4 h after administration of NNAL. (c) Each bar is the percent of total metabolites in a given tissue made up by a given metabolite 24 h after administration of NNAL. Tissues include liver (closed bars), lung (hatched bars) and kidney (open bars). Data presented as mean ± SD, n = 3; #, mean, n = 2. Note that the y-axis scale in (c) is different from (a,b). HA, hydroxy acid; KA, keto acid; LOX, NNAL-N-oxide; R-GL, (R)-NNAL-Gluc; UNK, unknown.
|
|
At 4 h after NNK dosing, NNK was observed in the liver (average 13.0% of total tissue metabolites, n = 2) and kidney (13.9%, n = 1) samples but not in the lung (Figure 4b
). NNAL was predominant in the lung (81.6 ± 14.4%) but relatively less abundant in the liver (30.2 ± 0.5%) and kidney (average 16.0%, n = 2). Hydroxy acid was not observed in the lung and liver but was present in the kidney. The abundance of NNAL (the parent compound) but lack of hydroxy acid (the metabolite) in the lung was particularly interesting because both NNAL and hydroxy acid were identified in the 1 h lung sample. (S)-NNAL was the predominant NNAL enantiomer in the lung sample at 4 h post-dose in contrast to the 1 h post-dose sample (Figure 5
).
The metabolite profiles in selected tissues were also evaluated after NNAL dosing. In the lung 1 h after racemic NNAL dosing, 48.6 ± 10.6% of the total metabolites was identified as NNAL, followed by NNAL-N-oxide (18.9 ± 10.3%), hydroxy acid (10.3 ± 2.4%) and keto acid (7.0 ± 0.4%) (Figure 6a
). In the liver, NNAL was found (52.8 ± 10.9% of total tissue metabolites, comparable with the lung), as well as significant amounts of (R)-NNAL-Gluc (13.4 ± 3.8%) and hydroxy acid (13.5 ± 10.5%). NNK was positively identified in the lung as well as liver samples, supporting the reversibility of its metabolism to NNAL. In the kidney, there were large amounts of NNAL (19.9 ± 13.7%), hydroxy acid (33.4 ± 12.0%) and keto acid (23.0 ± 5.9%). The kidney also had a relatively large amount of (R)-NNAL-Gluc (12.2 ± 4.7%), as high as that in the liver. The (S)/(R) ratio of NNAL was 0.35 ± 0.04 in the liver, 0.31 ± 0.11 in the lung and 0.19 ± 0.08 in the kidney (Figure 5
). At 1 h post-racemic NNAL dose, (R)-NNAL was clearly predominant over (S)-NNAL in all tissues.
At 4 h following the NNAL dose (Figure 6b
), NNAL remained the most abundant metabolite in the lung (42.8 ± 6.6% of tissue metabolites) but less was found in the liver (19.2 ± 4.8%) and kidney (19.2 ± 7.0%). (R)-NNAL-Gluc could still be detected in the liver and kidney at 4 h post-dose. The (S)/(R) ratios of NNAL shifted from those at 1 h (Figure 5
). A ratio of 2.3 ± 1.7 in liver samples and a ratio of 1.4 ± 0.4 in the lung samples was observed. Clearly at 4 h, the predominant form of NNAL in the lung and liver was (S)-NNAL.
This trend of (S)-NNAL predominance in tissue was strengthened by the samples taken 24 h post-dose (Figure 6c
). An (S)/(R) ratio of 57.3 (n = 2) in the lung, 1.2 (n = 2) in the liver and 3.4 (n = 2) in the kidney (Figure 5
) was observed. At 24 h, the keto acid was the only metabolite identifiable in all three tissues, and furthermore, made up the major proportion of metabolites in the liver and kidney. NNAL, however, was quantifiable only in the lung, and consisted of a striking 75.4% of total metabolites present in that tissue.
Figure 7
is a schematic of the stereoselective disposition of the NNAL enantiomers based on the present in vivo work as well as previous in vitro findings (18). Metabolism of NNK to (R)-NNAL appears to lead to detoxification through glucuronidation and biliary excretion. Metabolism of NNK to (S)-NNAL appears to favor
-hydroxylation as well as stereoselective localization in the lung.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7. Schematic of the stereoselective disposition of the NNAL enantiomers in rats based on the present in vivo work as well as previous in vitro findings (18). The larger arrows represent favored pathways.
|
|
 |
Discussion
|
---|
The first objective of the present study was to individually characterize the pharmacokinetic properties of NNK and its major metabolite NNAL. Evaluation of the kinetics of the NNK/NNAL pair was complicated by the reversible metabolism between the two, which challenged the traditional interpretation of pharmacokinetic parameters. Bearing this in mind, it is safe to say that NNK and racemic NNAL were compounds with very large volumes of disposition and low to intermediate total body clearances. The volumes of distribution and half-lives reported here are considerably greater than those reported earlier (16) for F344 rats. However, the reason for the apparent discrepancy lies with assay considerations. In both the present study and in the earlier report, the plasma half-life appeared to be very short. However, when evaluating the urinary excretion rate profiles, it is evident that the terminal elimination half-life is much longer than that observed in plasma. The plasma concentrations reach the lower limit of quantitation while the compounds are still in the distribution phase, and therefore the plasma half-lives cannot be considered to be the true elimination half-lives. For this reason, the terminal elimination half-lives from the urine were used to determine the `tail' portions of the AUC and AUMC. This is likely responsible for the larger volumes of distribution reported in the present study. As in the previous study, however, the volume of distribution of NNAL was greater than that of NNK, and its elimination half-life was longer (16). As also indicated by Adams et al., NNK was able to be formed from NNAL in vivo (16). The large volumes of distribution support the concept that NNK and NNAL are very widely distributed into the tissue (5).
The second objective of the study was to evaluate the patterns of biliary and urinary excretion of NNK, NNAL and their metabolites after individual administration of either NNK or NNAL. After 24 h, roughly 85% of a total dose of NNK (17.5 ± 3.8% from bile and 67.6 ± 10.1% from urine) was recovered. The extent of urinary excretion and urinary metabolite pattern were largely similar to previous NNK studies in rats with intact bile ducts (5,23), indicating that enterohepatic circulation of metabolites was probably unimportant. Approximately 60% of the total dose (19.5 ± 2.6% from bile and 42.6 ± 8.2% from urine) was recovered 24 h after NNAL administration. As previously reported from in vivo studies with NNK in the rat (6), NNAL-Gluc comprised the majority of the metabolites in the bile. The biliary excretion profiles after either NNK or racemic NNAL were very similar, with (R)-NNAL-Gluc comprising roughly 85% of the total metabolites excreted in the bile. The urinary and biliary excretion pattern of metabolites after administration of either compound were qualitatively similar, suggesting significant interconversion between NNK and NNAL.
A good marker for the extent of the re-conversion of NNAL to NNK in vivo might be the urinary excretion of keto acid. When NNK was administered, 26.0 ± 3.3% of the dose was excreted in the urine as the keto acid. When NNAL was administered, 9.18 ± 2.15% of the dose was excreted in the urine as keto acid. A previous study in rats had shown that keto acid was not formed in vivo from hydroxy acid (22) and therefore keto acid must arise from NNK after its formation from NNAL. This indicates that in vivo,
30% of a racemic NNAL dose is converted to NNK. Most of the NNK may come from (S)-NNAL (18) which would indicate that almost 60% of (S)-NNAL in the racemate was converted back to NNK. To directly examine whether the reversible metabolism of NNAL to NNK is stereoselective in vivo, each enantiomer should be administered individually. Those studies are currently in progress.
A previous study in the A/J mouse indicated that (S)-NNAL and NNK were equipotent in lung tumorigenicity, but (R)-NNAL was significantly less potent in the same bioassay (17). The carcinogenic difference of (S)- vs. (R)-NNAL could be caused by the stereoselective difference in the in vivo disposition profile of the two enantiomers rather than a difference in their intrinsic tumorigenic activity. A third objective of the present study was to determine the extent of stereoselective metabolism and disposition of NNAL and its metabolites in the F344 rat. Rats and mice do appear to have similar overall metabolic profiles after administration of NNK (3), and the urinary metabolite pattern is similar between rats and mice after racemic NNAL administration, except that mice were shown to excrete significantly more (R)-NNAL-Gluc in the urine (17) than did the rats in the present study. Therefore, it seems appropriate to evaluate stereoselective pharmacokinetic mechanisms in the F344 rat, namely stereoselective metabolism, distribution or transport.
Recent in vitro studies indicated that NNK was metabolized to (S)-NNAL to a much greater extent than to (R)-NNAL in both human and rodent tissues (18). Additionally in rat lung microsomes, (S)-NNAL was preferentially metabolized via
-hydroxylation over (R)-NNAL. Rat and human liver microsomes did not appear to show this stereoselectivity in metabolism (18). However, these in vitro studies were not optimized for formation of glucuronides. In the present in vivo studies, it was clearly shown that NNAL glucuronidation was a stereoselective process. In the NNAL study, (R)-NNAL-Gluc predominated in the bile although a racemic mixture of NNAL was administered. In addition, (R)-NNAL-Gluc was the only glucuronide metabolite observed in the liver. (R)-NNAL was the preferred form of NNAL in the liver during the first hour after NNAL administration. The (S)/(R) ratio of NNAL in the tissue at 1 h was
0.3 while a disproportionate amount of (R)-NNAL-Gluc (roughly a 1:100 ratio for (S)-/(R)-NNAL-Gluc, data not shown) was excreted in the bile. Approximately 4 h after administration, (S)-NNAL was the predominant form of NNAL in the liver, but (S)-NNAL-Gluc was not quantifiable in the tissue. At that time, (R)-NNAL-Gluc still predominated in the bile. This suggests that stereoselective formation of the glucuronide occurs rather than stereoselective secretion into the bile. In vivo, (R)-NNAL appeared to be much more rapidly eliminated, particularly because of (R)-NNAL-Gluc formation, compared with the (S)-enantiomer. This result and the stereoselectivity of (S)-NNAL
-hydroxylation in the lung may account for the findings in mice that the (S)-enantiomer is more carcinogenic (17).
However, stereoselective tissue distribution may also contribute to the relatively greater carcinogenic potential of (S)-NNAL. In as early as 5 min after NNK administration, the (S)/(R) ratio of NNAL in the plasma was close to unity, but decreased over time (Figure 3
) suggesting that (S)-NNAL was distributing to tissue more rapidly. In addition, after racemic NNAL administration, (S)-NNAL was the more long-lived enantiomer and was the predominant form found in the 24 h tissue samples. In the lung, it was found that regardless of the compound administered (NNK or NNAL), at later time points, (S)-NNAL was the predominant compound. Four hours after administration of NNK, (S)-NNAL made up 66% of total metabolites in the lung, with the (S)-enantiomer present in a ratio of 4:1 over the (R)-enantiomer. In the NNAL study, the overwhelming proportion of NNAL in the lung at 24 h was the (S)-enantiomer. This supports the hypothesis that (S)-NNAL was stereoselectively retained in the lung. In previous studies in the rat, NNK was administered either as a single injection of 1.76 mg/kg or as 1.76 mg/kg given three times weekly for 12, 24, or 36 doses (25). Tissue samples were taken at various times after the last dose. Of all the metabolites quantitated in the lung, only NNAL appeared to accumulate substantially with increasing numbers of doses (25). Although the enantiomers of NNAL were not separated in that study, the findings corroborate a long-term exposure of lung tissue to NNAL.
The mechanism of stereoselective localization of (S)-NNAL to the lung tissue is currently being evaluated. NNK has been shown to bind to nicotinic acetylcholine receptors in human lung cancer cells (26) as well as to ß1- and ß2-adrenergic receptors (27). NNK binding to ß1- and ß2-adrenergic receptors in a human adenocarcinoma cell line was associated with arachidonic acid release and increased DNA synthesis (27). The relative binding affinity of NNK, (S)-NNAL and (R)-NNAL for the ß-adrenergic receptors may provide some insight into the stereoselective binding of NNK metabolites to the lung tissue.
The finding that the more carcinogenic enantiomer of NNAL is selectively retained in lung tissue indicates a multi-factorial mechanism for lung carcinogenesis by NNK. Not only does the activation of NNK and NNAL to DNA-binding intermediates play a role in lung tumor initiation, but also the prolonged exposure to the more carcinogenic enantiomer (S)-NNAL. The stereoselective localization of (S)-NNAL to lung tissue may also contribute to the lung selectivity of NNK carcinogenesis. The present studies suggest that chemopreventive strategies should extend beyond single interventions such as inhibition of metabolic activation of NNK, and might include selective inhibition of (S)-NNAL binding to lung sites.
 |
Notes
|
---|
3 To whom correspondence should be addressed 
 |
Acknowledgments
|
---|
This study was supported by PHS grant CA-81301 from the National Cancer Institute. Stephen S.Hecht is an American Cancer Society Research Professor, supported by ACS grant RP-00-138.
 |
References
|
---|
-
Greenlee,R.T., Murray,T., Bolden,S. and Wingo,P.A. (2000) Cancer statistics, 2000. CA: Cancer J. Clin., 50, 733.[Abstract/Free Full Text]
-
Hecht,S.S. and Hoffmann,D. (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis, 9, 875884.[Abstract]
-
Hecht,S.S. (1998) Biochemistry, biology and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol., 11, 560603.
-
Belinsky,S.A., Foley,J.F., White,C.M. Anderson,M.W. and Maronpot,R.R. (1990) Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res., 50, 37723780.[Abstract]
-
Castonguay,A., Tjalve,H. and Hecht,S.S. (1983) Tissue distribution of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its metabolites in F344 rats. Cancer Res., 43, 630638.[Abstract]
-
Schulze,J., Richter,E., Binder,U. and Zwickenpflug,W. (1992) Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat. Carcinogenesis, 13, 19611965.[Abstract]
-
Carmella,S.G., Akerkar,S. and Hecht,S.S. (1993) Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers' urine. Cancer Res., 53, 721724.[Abstract]
-
Smith,T.J., Liao,A.M., Liu,Y., Jones,A.B., Anderson,L.M. and Yang,C.S. (1997) Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes. Carcinogenesis, 18, 15771584.[Abstract]
-
Staretz,M.E., Koenig,L.A. and Hecht,S.S. (1997) Effects of long term dietary phenethyl isothiocyanate on the microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats. Carcinogenesis, 18, 17151722.[Abstract]
-
Schrader,E., Hirsch-Ernst,K.I., Richter,E. and Foth,H. (1998) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver. Naunyn-Schmied. Arch. Pharmacol., 357, 336343.[ISI][Medline]
-
Carmella,S.G., Borukhova,A., Akerkar,S.A. and Hecht,S.S. (1997) Analysis of human urine for pyridine-N-oxide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific lung carcinogen. Cancer Epidemiol. Biomarkers Prev., 6, 113120.[Abstract]
-
Morse,M.A., Eklind,K.I., Toussaint,M., Amin,S.G. and Chung,F. (1990) Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its dose-dependent excretion in the urine of mice and rats. Carcinogenesis, 11, 18191823.[Abstract]
-
Hecht,S.S., Trushin,N., Reid-Quinn,C.A. et al. (1993) Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites. Carcinogenesis, 14, 229236.[Abstract]
-
Peterson,L.A., Mathew,R. and Hecht,S.S. (1991) Quantitation of microsomal
-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res., 51, 54955500.[Abstract]
-
Hecht,S.S., Spratt,T.E. and Trushin,N. (1997) Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis, 18, 18511854.[Abstract]
-
Adams,J.D., LaVoie,E.J. and Hoffmann,D. (1985) On the pharmacokinetics of tobacco-specific N-nitrosamines in Fischer rats. Carcinogenesis, 6, 509511.[Abstract]
-
Upadhyaya,P., Kenney,P.M.J., Hochalter,J.B., Wang,M. and Hecht,S.S. (1999) Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse. Carcinogenesis, 20, 15771582.[Abstract/Free Full Text]
-
Upadhyaya,P., Carmella,S.G., Guengerich,F.P. and Hecht,S.S. (2000) Formation and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers in vitro in mouse, rat and human tissues. Carcinogenesis, 21, 12331238.[Abstract/Free Full Text]
-
Hecht,S.S., Young,R. and Chen,C.B. (1980) Metabolism in the F344 rat of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Cancer Res., 40, 41444150.[Abstract]
-
Carmella,S.G., Ye,M., Upadhyaya,P. and Hecht,S.S. (1999) Stereochemistry of metabolites of a tobacco-specific lung carcinogen in smokers' urine. Cancer Res., 59, 36023605.[Abstract/Free Full Text]
-
Gibaldi,M. and Perrier,D. (1982) Pharmacokinetics, 2nd edn. Marcel Dekker, New York NY, pp. 409417.
-
Hecht,S.S., Lin,D. and Chen,C.B. (1981) Comprehensive analysis of urinary metabolites of N'-nitrosonornicotine. Carcinogenesis 9, 833838.
-
Desai,D., Kagan,S.S., Amin,S., Carmella,S.G. and Hecht,S.S. (1993) Identification of 4-(methylnitrosamino)-1-[3-(6-hydroxypyridyl)]-1-butanone as a urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rodents. Chem. Res. Toxicol., 6, 794799.[ISI][Medline]
-
Staretz,M.E. and Hecht,S.S. (1995) Effects of phenethyl isothiocyanate on the tissue distribution of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and metabolites in F344 rats. Cancer Res., 55, 55805588.[Abstract]
-
Schuller,H.M. and Orloff,M. (1998) Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells. Biochem. Pharmacol., 55, 13771384.[ISI][Medline]
-
Schuller,H.M., Tithof,P.K., Williams,M. and Plummer,H. III. (1999) The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a ß-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via ß-adrenergic receptor-mediated release of arachidonic acid. Cancer Res., 59, 45104515.[Abstract/Free Full Text]
Received June 29, 2001;
revised September 25, 2001;
accepted October 9, 2001.