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Carcinogenesis Advance Access originally published online on August 27, 2007
Carcinogenesis 2007 28(12):2650-2656; doi:10.1093/carcin/bgm187
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Biotransformation and transport of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in bile duct-cannulated wild-type and Mrp2/Abcc2-deficient (TR–) Wistar rats

Elaine M. Leslie1,2, Giulia Ghibellini1,3, Ken-ichi Nezasa1,4 and Kim L.R. Brouwer1,*

1 School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
2 Present address: Membrane protein research group Department of Physiology, University of Alberta, Edmonton, AB T6G 2H7, Canada
3 Present address: Clinical Pharmacology and Discovery Medicine, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
4 Present address: Development Research Laboratories, Shionogi & Co., Ltd, Toyonaka, Osaka 561-0825, Japan

* To whom correspondence should be addressed. Tel: +1 919 962 7030; Fax: +1 919 966 0197; Email: kbrouwer{at}unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The role of uptake and efflux transport proteins in the tissue distribution of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites is largely unknown. Carbonyl reduction of NNK results in formation of the carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which in rats is glucuronidated to the non-toxic NNAL-O-glucuronide. Previous in vitro studies showed that NNAL-O-glucuronide is a substrate for the human ATP-binding cassette transport proteins multidrug resistance protein (MRP)1 (ABCC1) and MRP2 (ABCC2). To investigate the influence of Mrp2 deficiency on NNK biotransformation and biliary excretion, [3H]NNK was administered intravenously to bile duct-cannulated wild-type (WT) and Mrp2-deficient (TR–) Wistar rats; plasma, bile and urine samples were collected for 5 h and analyzed by high-pressure liquid chromatography with radiochemical detection. The total radioactivity recovered in WT and TR– bile was 12 and 7% of the dose, respectively. NNAL-O-glucuronide accounted for 87% of the radioactivity in WT bile but was not detected in TR– bile. Urinary recovery of 1-(3-pyridyl)-1-butanol-4-carboxylic acid (hydroxy acid), NNAL-O-glucuronide and NNAL-N-oxide from 2–5 h was greater in TR– compared with WT rats. NNK plasma clearance was significantly higher in TR– (115 ± 23 ml/min/kg) compared with WT (48 ± 13 ml/min/kg) rats. A higher concentration and/or earlier appearance of hydroxy and 1-(3-pyridyl)-1-butanone-4-carboxylic acids, NNAL-N-oxide and NNK-N-oxide, and decreased NNK and NNAL concentrations in TR– plasma suggested increased cytochrome P450 biotransformation in TR– rats. The total recovery of hydroxy acid in bile and urine was significantly higher in TR– compared with WT rats. Thus, Mrp2 is responsible for the biliary excretion of NNAL-O-glucuronide and Mrp2 deficiency results in increased formation of carcinogenic NNK metabolites.

Abbreviations: ABC, ATP-binding cassette; CL, clearance; CLbiliary, biliary clearance; CLrenal, renal clearance; CYP450, cytochrome P450; HPLC, high-pressure liquid chromatography; hydroxy acid, 1-(3-pyridyl)-1-butanol-4-carboxylic acid; keto acid, 1-(3-pyridyl)-1-butanone-4-carboxylic acid; MRP, multidrug resistance protein; MW, molecular weight; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; WT, wild-type


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The nicotine-derived tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most prevalent and toxic procarcinogens found in tobacco products (1,2). Carbonyl reduction of NNK results in the formation of a second procarcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which can be subsequently glucuronidated to the non-toxic metabolites NNAL-O-glucuronide or the more recently identified NNAL-N-glucuronide (3,4). Rodents metabolize NNAL primarily to (R)NNAL-O-glucuronide, whereas (S)- and (R)-NNAL-O-glucuronide and NNAL-N-glucuronide have been identified in human urine (3,5). Pyridine N-oxidation of NNK and NNAL results in the formation of other detoxification products, NNK-N-oxide and NNAL-N-oxide (Figure 1). Bioactivation of NNK and NNAL, predominantly through cytochrome P450 (CYP450) pathways, results in hydroxylation of the methyl and methylene carbons adjacent to the N-nitroso group and leads to the formation of methyl and pyridyloxobutyl DNA adducts (1,6,7).


Figure 1
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  		Fig. 1. Primary biotransformation pathways of NNK in rats. Modified from ref. (8).

 
In rodent models, the lung-specific carcinogenicity of NNK is remarkably independent of the administration route, and thus far it appears that this tissue-selective toxicity is related to tissue-specific P450-dependent bioactivation (1,7). However, the role of uptake and efflux transport proteins in the distribution of NNK and its metabolites is largely unexplored. NNAL is stereoselectively metabolized and excreted; (S)-NNAL is retained unchanged in the lung, whereas (R)-NNAL enters detoxification pathways more easily. In rats, significantly more (R)-NNAL-O-glucuronide is excreted into bile than the (S)-glucuronide (8,9). NNK metabolism is likely the rate-limiting step for excretion; however, tissue differences in transport protein expression could alter the cellular uptake and retention of NNK/metabolites and affect tissue susceptibility to carcinogenic effects.

Several members of the ATP-binding cassette (ABC) transporter superfamily have important roles in xenobiotic and/or metabolite elimination and are therefore of critical importance for cellular and tissue defense (10). Previous in vitro studies have shown that (R)-NNAL-O-glucuronide is a substrate for two such ABC transport proteins, the multidrug resistance protein (MRP)1 (ABCC1) and MRP2 (ABCC2) (11). MRP2 is expressed on the apical membrane of polarized cells, including hepatocytes, renal proximal tubular cells, enterocytes and placental syncytiotrophoblasts (12,13). The substrate specificity and cellular/tissue localization of MRP2 suggests that it is important for the excretion of endogenous and xenobiotic compounds, particularly substances that undergo glutathione, glucuronide or sulfate conjugation (13,14).

Genetic deficiency in MRP2 results in Dubin–Johnson syndrome in humans, a form of congenital hyperbilirubinemia. This hyperbilirubinemia is caused by an increased efflux of bilirubin-glucuronide from hepatocytes into blood by the related basolateral transporter MRP3/ABCC3, which is induced in the absence of MRP2 (13). The phenotypes of Mrp2-deficient rats, TR– and Eisai hyperbilirubinemic, are similar to Dubin–Johnson syndrome and these rat models have been useful in determining the substrate specificity and in vivo function of both MRP2 and MRP3 (15–17).

In vitro evidence combined with Mrp2 localization and substrate specificity suggests that Mrp2 is a likely candidate transport protein responsible for the extensive biliary excretion of NNK metabolites, in particular NNAL-O-glucuronide, that occurs after NNK administration to rats (8,9,18). In the present study, the effects of Mrp2 deficiency on the metabolism and disposition of NNK were investigated. Anesthetized bile duct-cannulated male wild-type (WT) and TR– Wistar rats were administered a single bolus dose of [3H]NNK and serial plasma, bile and urine samples were collected for 5 h and analyzed by high-pressure liquid chromatography (HPLC) with radiochemical detection. Mrp2 was shown to be critical for biliary excretion of NNAL-O-glucuronide. Results also suggested that there was a shift in the NNK metabolic pathway between TR– and WT rats, which resulted in an increased plasma clearance (CL) of NNK in TR– rats consistent with elevated formation of CYP450-derived metabolites.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals and reagents
[3H]NNK (11 Ci/mmol; 99% purity) was purchased from Moravek Biochemicals (Brea, CA). NNK, NNAL, 1-(3-pyridyl)-1-butanol-4-carboxylic acid (hydroxy acid), 1-(3-pyridyl)-1-butanone-4-carboxylic acid (keto acid), NNK-N-oxide and NNAL-N-oxide were purchased from Toronto Research Chemicals (North York, Ontario, Canada). FLO-SCINT III was purchased from PerkinElmer (Boston, MA). β-Glucuronidase type B3 from bovine liver was purchased from Sigma (St Louis, MO).

Animals
Male Wistar rats (200–310 g, Charles River Laboratories, Raleigh, NC) or male Mrp2-deficient TR– rats bred in our animal facility (195–300 g, breeders were originally obtained from Dr Mary Vore, University of Kentucky, Lexington, KY) were maintained on a 12 h light–dark cycle with access to water and rodent chow ad libitum. Rats were allowed to acclimate for at least 5 days before experimentation.

Animal studies
Anesthesia was induced intra-peritoneally with 60 mg/kg ketamine and 12 mg/kg xylazine (Vedco, St Joseph, MO) and maintained with 10 mg/kg ketamine and 2 mg/kg xylazine every 60–90 min. When given in combination, ketamine and xylazine have no effect on bile or urine flow in rats (19,20). The right jugular vein, common bile duct and urinary bladder were cannulated with polyethylene tubing; PE-50, PE-10 and PE-50, respectively (Becton Dickinson, Parsippany, NJ).

The dosing solution for each rat was prepared in saline by diluting 10 µCi [3H]NNK with unlabeled NNK to obtain a total dose of 0.7 µmol/kg. The dose was given through the caudal vein as a bolus and each rat received ~200 µl of dosing solution.

Blood samples (200 µl) were collected at 5, 15, 30, 60, 120, 180, 240 and 300 min after dose from the jugular cannula using a heparinized 1 ml syringe. After each collection, the cannula was flushed with 200 µl of saline. Blood was centrifuged at 10 000g for 5 min and plasma was collected. Urine was collected over two time intervals, 0–2 and 2–5 h after dose. Bile was collected at the following intervals after dose: 0–15, 15–30, 30–60, 60–90, 90–120, 120–180, 180–240 and 240–300 min. Aliquots of plasma, urine and bile samples (75, 100 and 10 µl, respectively) were subjected to liquid scintillation counting and the remainder of the sample was stored at –80°C until HPLC analysis.

Analysis of NNK and metabolites
Samples were separated by reverse-phase HPLC (HP1050, Agilent Technologies, Waldbronn, Germany) fitted with an ultraviolet–visible detector operated at {lambda} 254 nm and a Flo-one β-radioactivity flow detector (500 TR, PerkinElmer, Waltham, MA). Bile and urine samples were centrifuged for 10 min at 16 100g and then 50–100 µl of sample was analyzed. Plasma was diluted 1:1 with ice-cold acetonitrile, vortexed, centrifuged for 10 min at 16 100g and the supernatants were evaporated to dryness under N2. The residue was reconstituted in 80 µl of 20 mM Na2HPO4, pH 6, and 70 µl was analyzed by HPLC. Bile was analyzed using an Aquasil column (150 x 2 mm, 5 µm, C18; Allied Biosystems, Bellefonte, PA) at 25°C with a flow rate of 0.5 ml/min; 100% mobile phase A (20 mM Na2HPO4, pH 6) was held for 5 min followed by a 20 min gradient to 76% mobile phase A/24% mobile phase B (100% acetonitrile), followed by a 2 min gradient to 100% mobile phase A and a 5 min hold at 100% mobile phase A. Plasma and urine samples were analyzed using a Licrospher 60 RP-selectB column (250 x 4 mm, 5 µm, C18; Agilent Technologies), which gave better resolution of the peaks in these matrices. Analysis was done at 25°C with a flow rate of 1 ml/min; 95% mobile phase A/5% mobile phase B was held for 10 min followed by a gradient to 70% mobile phase A/30% mobile phase B over 30 min, followed by a 2 min gradient to 50% mobile phase A/50% mobile phase B, followed by a 2 min gradient to 95% mobile phase A/5% mobile phase B and 6 min at 95% mobile phase A/5% mobile phase B.

Radioactive metabolites were confirmed by co-chromatography with a mix of non-radiolabeled reference compounds (hydroxy acid, keto acid, NNAL-N-oxide, NNK-N-oxide, NNAL and NNK) detected by ultraviolet absorbance. The presence of NNAL-O-glucuronide was confirmed by treatment of samples with β-glucuronidase type B3 from bovine liver (1000 U) at 37°C for 24 h at pH 7.

Metabolite and parent concentrations in each sample were calculated as described previously (9). Briefly, total radioactivity ([3H]NNK and [3H]metabolites) in each sample was quantified by liquid scintillation counting, and the concentration of total NNK and metabolites was calculated from the original specific activity. Samples were then separated by HPLC, the peak areas of individual metabolites and NNK were determined and expressed as a ratio of the integrated area under each peak divided by the total integrated area of the HPLC chromatogram. Concentrations of each analyte were determined by multiplying the total concentration (determined above) by the percentage of total radioactivity in each peak to obtain the total counts for each compound. This was then converted to the concentration of each compound (expressed in terms of nanomolar of NNK equivalents) with the use of the original specific activity.

Pharmacokinetic analysis
Non-compartmental analysis was used to calculate pharmacokinetic parameters using WinNonlin Pro version 4.2 (Pharsight, Mountain View, CA) assuming linear pharmacokinetics with unidirectional metabolic pathways. The area under the plasma concentration–time curve (AUC) for NNK was calculated using the linear trapezoidal rule and extrapolated to infinity. NNK plasma CL, biliary clearance (CLbiliary) and renal clearance (CLrenal) were calculated as follows:

Formula

Formula

Formula
where X0 was the dose (µmol/kg) administered via the caudal vein and X was the amount of NNK in the designated sample. In all cases, the extrapolated AUC from T300 min to infinity was <20% of the total AUC.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Biliary excretion of NNK and metabolites by WT and TR– Wistar rats
The excretion of [3H]NNK and metabolites was studied in bile duct-cannulated TR– and WT rats that maintained bile flow values greater than ~60% of baseline bile flow throughout the collection period. As expected, bile flow in TR– rats was reduced to ~65% of WT levels (Figure 2A) (21). When cumulative biliary excretion of total radioactivity was measured in bile from WT and TR– rats, significantly more radioactivity was detected in WT bile (Figure 2B and C). The cumulative biliary excretion profile in WT rats approached a maximal value of 12% of the NNK dose at 300 min (Figure 2B). HPLC analysis of WT bile revealed that NNAL-O-glucuronide accounted for 87% of total biliary radioactivity, while minor metabolites; NNAL (~5%), keto acid (~4%), hydroxy acid (~2%) and parent NNK (~3%) accounted for the remaining radioactivity (Figure 2B and D). The identity of the NNAL-O-glucuronide was confirmed by treating bile samples with β-glucuronidase and analyzing them by HPLC. This resulted in the complete loss of the peak presumed to be NNAL-O-glucuronide and the appearance of a peak at the same retention time as NNAL (data not shown). Total radioactivity in TR– bile increased in a linear fashion through 300 min, accounting for 7.6% of the NNK dose. HPLC analysis of TR– bile revealed that hydroxy acid accounted for 57% of the total radioactivity (Figure 2D), with NNAL-N-oxide (23%), NNAL (10%), keto acid (5%) and NNK (3%) accounting for the remainder of the total radioactivity. NNAL-O-glucuronide was not detected in TR– bile. The CLbiliary of the parent compound (NNK) was not significantly different between the two rat strains (Table I).


Figure 2
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  		Fig. 2. Excretion of NNK and metabolites into the bile of WT and TR– bile duct-cannulated, anesthetized rats. [3H]NNK (0.7 µmol/kg, 10 µCi) was administered to WT and TR– rats through the caudal vein (three rats per group). (A) Bile flow was determined by collecting bile from WT (closed squares) (liver weights 7–14 g) and TR– (open squares) (liver weights 9–14 g) rats at the indicated time points, followed by gravimetric analysis. Cumulative biliary excretion of NNK and metabolites in (B) WT and (C) TR– rats was measured for 300 min after [3H]NNK administration. Samples were analyzed by liquid scintillation counting for total radioactivity (closed squares). Metabolites [hydroxy acid (open squares), keto acid (closed inverted triangles), NNAL-O-glucuronide (open squares), NNAL-N-oxide (open diamonds), NNAL (open circles) and NNK (open triangles)] were identified and quantitated by HPLC as described in Materials and Methods. (D) Total biliary recovery of NNK and metabolites in bile from WT (closed bars) and TR– (open bars) rats was calculated from total radioactivity determined by liquid scintillation counting and metabolite profiles determined by HPLC. Symbols and bars represent mean (± SD). *P < 0.05 WT versus TR–; Student's two-tailed t-test.

 


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  		Table I. Summary of NNK pharmacokinetic parameters

 
Urinary excretion of NNK and metabolites by TR– and WT Wistar rats
The predominant metabolites recovered in WT urine (total 0–5 h) were the keto acid (14% of dose), hydroxy acid (12% of dose) and NNAL-N-oxide (11% of dose). Other metabolites including NNK-N-oxide (5% of dose), an unknown metabolite (3.8% of dose), NNAL-O-glucuronide (1.5% of dose), NNAL (1.1% of dose) and NNK (0.3% of dose) also were present. The unknown compound had the same retention order as the keto alcohol, reported previously (9), for which a standard is not commercially available. The predominant metabolites found in TR– urine (total 0–5 h) were similar to WT animals: keto acid (20% of dose), hydroxy acid (20% of dose) and NNAL-N-oxide (14% of dose). NNK-N-oxide (5% of dose), an unknown metabolite (4% of dose), NNAL-O-glucuronide (3.8% of dose), NNAL (0.91% of dose) and NNK (0.22% of dose) accounted for the remainder of the recovered radioactivity. No significant difference was observed between total radioactivity recovered or the metabolite profile in urine from TR– and WT rats collected over the first 2 h after the NNK dose (Figure 3A). However, recovery of the hydroxy and keto acids, NNAL-O-glucuronide and NNAL-N-oxide was significantly higher in TR– urine collected over the 2–5 h time interval after dose (Figure 3B). Although there was a trend in TR– rats toward a higher NNK CLrenal (Table I) and total dose excreted in urine (Table II), these differences failed to reach statistical significance.


Figure 3
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  		Fig. 3. Urinary recovery of NNK and metabolites in WT and TR– bile duct-cannulated, anesthetized rats. [3H]NNK (0.7 µmol/kg and 10 µCi) was administered to WT and TR– rats (three rats per group) through the caudal vein. Urine was collected for (A) 0–2 h and (B) 2–5 h from the WT (closed bars) and the TR– (open bars) rats. NNK and metabolites were quantified (mean ± SD) using HPLC β-flow analysis as described in Materials and Methods. *P < 0.05 WT versus TR–; Student's two-tailed t-test.

 


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  		Table II. Total recovery of NNK and metabolites in bile and urine of WT and TR– rats

 
Analysis of NNK metabolites in plasma of TR– and WT Wistar bile duct-cannulated rats
The mean plasma concentration–time profiles for total radioactivity, NNK and NNK metabolites are shown for WT and TR– rats in Figure 4. The initial plasma concentrations based on total radioactivity (Figure 4A) were similar for both WT and TR– rats, confirming that the animals received similar doses. In both rat strains, NNK rapidly disappeared from the blood and was not detected in plasma 30 min after dose (Figure 4B). NNK plasma concentrations declined more rapidly in TR– rats. The 2.4-fold increase in NNK plasma CL (115 ± 23 versus 48 ± 13 ml/min/kg) (Figure 4B and Table I) in TR– rats with no change in CLbiliary or CLrenal of NNK suggests that the metabolic CL of NNK was increased in TR– rats.


Figure 4
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  		Fig. 4. Plasma profiles of NNK and metabolites in WT and TR– bile duct-cannulated, anesthetized rats. [3H]NNK (0.7 µmol/kg and 10 µCi) was administered to WT and TR– rats (three rats per group) through the caudal vein. (A) Plasma samples were collected at the indicated time points and total radioactivity quantitated by liquid scintillation counting for WT (closed squares) and TR– (open squares) rats. Plasma samples were analyzed by HPLC with radiochemical detection, allowing the identification and quantification of NNK and metabolites. Data represent plasma concentration (mean ± SD) versus time profiles of (B) NNK, (C) NNAL, (D) keto acid, (E) NNAL-N-oxide and (F) hydroxy acid in WT (closed symbols) and TR– (open symbols) rats.

 
Four NNK metabolites were detected in WT plasma with the following rank order of AUC0–300 min values: NNAL > keto acid > NNAL-N-oxide > hydroxy acid. In contrast, six metabolites were detected in TR– plasma with the following rank order of AUC0–300 min values: NNAL > keto acid ≥ hydroxy acid ≥ NNAL-N-oxide > NNK-N-oxide > unknown. In TR– rats, the plasma concentration of the keto acid was higher at the 5-min time point (Figure 4D), and NNAL-N-oxide and hydroxy acid were detected in TR– plasma at earlier time points compared with WT plasma (5 versus 30 min) (Figure 4E and F). In contrast to WT plasma, NNK-N-oxide and the same unknown metabolite detected in urine were present in TR– plasma between 15–30 and 5–15 min, respectively (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
NNK is a potent carcinogenic component of tobacco products, and also is found in environmental tobacco smoke and the urine of non-smokers exposed to second-hand smoke (22). Thus, understanding how NNK and its metabolites are formed, distributed and excreted is important for both tobacco product users as well as the population exposed passively to tobacco smoke. The role of uptake and efflux transport proteins in the tissue distribution of NNK and its metabolites has been largely unexplored. Evidence from in vivo studies indicates that NNK is rapidly taken up into the body, distributed and metabolized (8,9,18). Previous reports suggested that membrane passage of NNK occurs through passive diffusion (18,23). Preliminary investigations in our laboratory found that cellular uptake of NNK by primary hepatocytes occurred at the same rate at 4 and 37°C, consistent with passive transport across cell membranes (data not shown). Considering the neutral net charge, a LogD of 0.09, and the small molecular weight (MW) of NNK, this seems to be a reasonable conclusion (Table III). For the same reasons, NNAL probably traverses cellular membranes through a passive process (Table III).


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  		Table III. Physicochemical properties of NNK and metabolites

 
The passive nature of NNK and NNAL distribution suggests that uptake transport is not likely to be a factor in the tissue distribution of these carcinogens. However, transport of NNK and NNAL metabolites may be important for elimination and toxicity related to these compounds. Using in vitro assays, it was shown previously that the human ABC transport proteins MRP1 and MRP2 could transport NNAL-O-glucuronide (11). The transport of the negatively charged and bulkier NNAL-O-glucuronide from hepatocyte to bile was predicted to be an active process (18). The current study is the first in vivo investigation that identifies Mrp2 as the transport protein responsible for the biliary excretion of NNAL-O-glucuronide in the rat.

Considerable species differences exist in the excretion pattern of NNK metabolites. In general, urinary excretion of NNAL + NNAL-glucuronides is much higher in humans and other primates (20–100% of dose) than in rats (not detectable at low doses) (4,24,25), while biliary excretion of NNAL-O-glucuronide is much greater in rats (7–18%) than primates (0.62%) (9,18). In general, species differences exist in biliary excretion with a threshold MW of ~325 for biliary excretion in rats and ~500 in humans (26,27). Consistent with the MW threshold hypothesis, the relatively small NNAL-O-glucuronide (MW 385) would be excreted more likely in rat bile than human bile. Substantial evidence has accumulated to suggest that these species differences in biliary excretion may be related, in part, to species differences in Mrp2/MRP2 expression and function. Substrate specificities for Mrp2/MRP2 appear to be similar, but differences exist in expression levels (more extensive canalicular Mrp2 expression in rat), transport modulation, substrate interaction, affinity and efficiency (28–32). Thus, since NNK is excreted into rat bile exclusively by Mrp2, species differences in Mrp2/MRP2 probably explain the low biliary excretion of NNK metabolites in primates compared with the higher biliary excretion in rats.

In addition to Mrp2, it is important to consider the contribution of related sinusoidal/basolateral hepatic ABC transport proteins to NNK metabolite elimination. Basolateral hepatic transport proteins such as Mrp3 and Mrp4/Abcc4 are expressed on the membrane of the hepatocyte opposite to canalicular Mrp2, and transport metabolites from the hepatocyte to blood for renal excretion (10,33). NNAL-O-glucuronide was recovered in the urine of WT and TR– rats (Figure 3), suggesting that not all of this glucuronide conjugate formed in the liver is excreted in bile; some is actively transported into blood and then eliminated by the kidney. While extra-hepatic NNAL-O-glucuronide could be formed, this is unlikely, due to the exclusive hepatic expression of UDP glucuronosyltransferase 2B1, the rat enzyme most important for NNAL-O-glucuronide formation (34,35). Undoubtedly, Mrp3 is important for the hepatic basolateral export of NNAL-O-glucuronide, consistent with the finding that treatment of male F344 rats with phenobarbital, which is a known inducer of rat Mrp3 and UDP glucuronosyltransferases, resulted in a 4.6-fold increase in the urinary excretion of NNAL-O-glucuronide (36–39). In the present study, the well-characterized up-regulation of Mrp3 expression in livers of Mrp2-deficient rats (37,40,41) probably contributed to the 2-fold increase in urinary recovery of NNAL-O-glucuronide in TR– compared with WT rats (Figure 3).

The mean NNK plasma CL of 48 ml/min/kg (11.8 ml/min) determined in WT Wistar rats in the present study was nearly identical to the previously published values (12.8 ml/min in non-anesthetized bile duct-cannulated Fisher 344 rats) (9). NNK plasma CL was significantly increased in TR– compared with WT rats. When administered as the preformed metabolite, <5% of (R)-NNAL is excreted as NNK and NNK metabolites; in contrast, when (S)-NNAL is administered to rats, ~20% of the dose is excreted as NNK and NNK metabolites (8). Data analyses in the present study assumed that metabolism of NNK to NNAL was irreversible (as detailed in the Materials and Methods). If this assumption was not correct, the NNK AUC would represent both administered NNK and NNK generated from NNAL.

Interestingly, the total recovery of NNAL-O-glucuronide was 4-fold lower in TR– compared with WT rats (~3 versus 12% of dose, respectively). Residual NNAL-O-glucuronide was not detected in liver tissue from either WT or TR– rats at the end of the experiment (data not shown). UDP glucuronosyltransferase expression levels in TR– rats are known to be increased or equivalent to WT rats (42–44). These findings would suggest that alternate metabolic pathways are dominant in TR– rats. Increased NNK plasma CL, but not NNK CLrenal or CLbiliary, and increased recovery of hydroxy acid with a trend toward increased recovery of keto acid and NNAL-N-oxide in TR– compared with WT rats in the present study support previously published literature that differences exist in the CYP450 expression and activity profiles between the WT and TR– rats (42,45). Hepatic expression and/or activity of CYP450 2E1, 3A1/23, 1A1/2, 2B1/2, 2C11 and 2A11 were increased in male TR– compared with WT rats (42,45). In addition, total CYP450 and NADPH–P450 reductase activities were increased in liver microsomes prepared from male TR– versus WT rats (45). CYP450 isoforms that are thought to be important for NNK bioactivation in the rat include CYP450 2A3, 1A2, 1A1, 2C6 and 2B1 (7). Thus, since several of these enzymes are known to be elevated in TR– rats, this is a plausible explanation for the observation that more CYP450-mediated metabolites were detected in TR– plasma, metabolite concentrations appeared at earlier time points than in WT rats and NNK plasma CL was higher in TR– rats.

In summary, the current data clearly demonstrate that Mrp2 is responsible for the biliary excretion of NNAL-O-glucuronide. TR– rats exhibit increased NNK plasma CL and increased recovery of P450-derived metabolites including bioactivated compounds, which suggests increased mutagenic risk. Human and rat MRP2/Mrp2 have similar physiological functions, and it is possible that patients with Dubin–Johnson syndrome and other less severe ABCC2 polymorphisms (reviewed in ref. 13) may exhibit a similar shift in CYP450 gene expression and function. The overall consequences of such MRP2/Mrp2 mutations for cancer risk from NNK exposure are unknown, but deserve further investigation.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (GM41935); Canadian Institutes of Health Research Fellowship to E.M.L.; American Foundation for Pharmaceutical Education Fellowship to G.G.


   Acknowledgments
 
The authors would like to thank Terence Hill for technical assistance with HPLC analysis. Dr Arlene Bridges and Dr Bob St Claire are gratefully acknowledged for expert advice on HPLC methodology.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received February 27, 2007; revised July 13, 2007; accepted August 13, 2007.


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