(NNK) in isolated rat lung and liver - Springer Link

Naunyn-Schmiedeberg’s Arch Pharmacol (1998) 357 : 336–343

© Springer-Verlag 1998

O R I G I N A L A RT I C L E

E. Schrader · K. I. Hirsch-Ernst · E. Richter · H. Foth

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver

Received: 13 March 1997 / Accepted: 21 November 1997

Abstract The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects NNK requires metabolic activation which is supposed to take place via α-hydroxylation, whereas Noxidation is suggested to be a detoxification pathway. The differences in the organ specific metabolism of NNK may be crucial for the organotropy in NNK-induced carcinogenesis. Therefore, metabolism of NNK was investigated in the target organ lung and in liver of Fischer 344 (F344) rats using the model of isolated perfused organs. High activity to metabolize 35 nM [5-3H]NNK was observed in both perfused organs. NNK was eliminated by liver substantially faster (clearance 6.9 ± 1.6 ml/min, half-life 14.6 ± 1.2 min) than by lung (clearance 2.1 ± 0.5 ml/min, halflife 47.9 ± 7.4 min). When the clearance is calculated for a gram of organ or for metabolically active cell forms, the risk with respect to carcinogenic mechanisms was higher in lung than in liver. The metabolism of NNK in liver yielded the two products of NNK α-hydroxylation, the 4-oxo-4-(3-pyridyl)butyric acid (keto acid) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid). In lung, the major metabolite of NNK was 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)1-butanone (NNK-N-oxide). Substantial amounts of metabolites formed from methyl hydroxylation of NNK, which is one of the two possible pathways of α-hydroxylation, were detected in lung but not in liver perfusion. Formation of these metabolites (4-oxo-4-(3-pyridyl)-butanol

E. Schrader · K. I. Hirsch-Ernst Institute of Toxicology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany E. Richter Walther-Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University München, Nußbaumstrasse 26, D-80336 München, Germany H. Foth (Y) Institute of Environmental Toxicology, Martin Luther University Halle-Wittenberg, Franzosenweg 1 a, D-06097 Halle/Saale, Germany

(keto alcohol), and 4-hydroxy-4-(3-pyridyl)-butanol (diol) can give rise to pyridyloxobutylating of DNA. When isolated rat livers were perfused with 150 µM NNK, equal to a dosage which is sufficient to induce liver tumors in rat, glucuronidation of 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL) was increased when compared to the concentration of 35 nM NNK. Nevertheless, the main part of NNK was also transformed via α-hydroxylation for this high concentration of NNK. Key words NNK · Elimination kinetics · Metabolism · Perfusion · Lung · Liver · Rat · N-oxide

Introduction Cigarette smoking has been demonstrated to be a major risk factor for lung cancer (Peto et al. 1992). Tobacco smoke contains about 50 identified carcinogens including the nicotine derived 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK), one of the most potent tobacco-specific carcinogens (Hecht et al. 1978; Brunnemann and Hoffmann 1993) which is thought to be the major cause of pulmonary adenocarcinoma in cigarette smokers (Hoffmann et al. 1994). In rodent species NNK shows dose dependent activity to induce tumors with a different incidence between target tissues, such as lung and pancreas. At low doses NNK induces primarily lung tumors in rats (Hoffmann et al. 1984) as well as in mice and hamsters independent of the route of administration (Hoffmann et al. 1994). However, it remains unclear what are the causes for the sensitivity of lung to develop adenoma and adenocarcinoma after NNK exposition. Like other procarcinogens NNK requires metabolic activation to elicit its tumorigenic effects. Whereas metabolic activation of NNK has been demonstrated in lung tissue explants, isolated cells and microsomes from a variety of species, including hamster, mouse, rat and man (Tjälve 1991, for review), only little is known about NNK metabolism in the intact lung where the structural features of the organ are taken into account (Foth 1995; Foth et al.

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1991). According to Rivenson et al. (1991), NNK may be considered a ‘blood-borne’ carcinogen because it is transported by the blood stream via the vascular network to the individual organs and tissues like the liver and nasal cavity. This route of exposure may also target the lung apart from inhalation. Therefore, vascular perfusion of isolated organs may be a suitable model to study the fate of NNK in its major target organ lung, providing intact architectural features. The suspected metabolic pathways are keto reduction of NNK to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N-oxidation of NNK and NNAL to 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-Noxide) and 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)1-butanol (NNAL-N-oxide), and α-hydroxylation of both NNK and NNAL, yielding 4-oxo-4-(3-pyridyl)-butanol (keto alcohol), 4-hydroxy-4-(3-pyridyl)-butanol (diol), 4oxo-4-(3-pyridyl) butyric acid (keto acid) and 4-hydroxy4-(3-pyridyl)-butyric acid (hydroxy acid). NNAL is a substrate of glucuronyl transferase, leading to the formation of one (rats, mice) or two (patas monkey, humans) stereo isomers of [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]β-O-D-glucopyranosiduronic acid (NNAL-Glu) (Morse et al. 1990; Hecht et al. 1993 b; Murphy et al. 1994). Ring hydroxylation of NNK has also been confirmed as an additional minor metabolic pathway (Desai et al. 1993). The pattern of metabolites obtained after incubation of NNK with isolated cells or subcellular fractions in vitro differs substantially from that formed in vivo. Rat lung cells incubated with 50 µM NNK release mainly NNAL into the medium (Belinsky et al. 1989). NNAL is also the major metabolite of NNK in lung microsomes of hamsters (Jorquera et al. 1993). The rates of α-hydroxylation, Noxidation and keto reduction are almost the same in mouse and rat lung microsomes incubated with NNK at or below a concentration of 10 µM (Smith et al. 1990; Hong et al. 1992; Guo et al. 1992 b, 1993). In human lung microsomes, NNAL accounts for > 95% of all metabolites (Smith et al. 1992). In rat hepatocytes (freshly isolated or in culture) NNK and NNAL is primarily metabolized via α-hydroxylation, whereas formation of N-oxides is only a minor pathway (Liu et al. 1990; Alaoui-Jamali et al. 1991). Experiments on NNK metabolism in liver microsomes have shown that NNAL is quantitatively the main metabolite in rat, hamster as well as in human liver (Guo et al. 1992 a; Jorquera et al. 1992; Smith et al. 1992). These differences between the urinary metabolite pattern of NNK and the results obtained by various in vitro methods prompted us to investigate the metabolism of NNK in the isolated perfused rat lung and liver. This method has been used to demonstrate the influence of phenobarbitone and other xenobiotics on the kinetics and metabolism of other compounds of cigarette smoke, such as nicotine (Foth et al. 1991) and benzo[a]pyrene (Foth et al. 1984). The aim of the present study was therefore to compare NNK kinetics and metabolism in perfused lung as the preferential target organ of NNK-induced carcinogenesis to kinetics and metabolism in perfused liver, particularly with regard to the organotropic effects of NNK.

Materials and methods Chemicals. [5-3H]NNK with a specific activity of 2.89 Ci/mmol, was obtained from Campro Scientific (Emmerich, Germany). HPLC analysis confirmed > 98% radiochemical purity. Unlabelled reference compounds for known metabolites of NNK were a generous gift from Dr. D. Hoffmann (American Health Foundation, Valhalla, N.Y., USA). HPLC-grade solvents were obtained from Merck (Darmstadt, Germany). All other chemicals were of the highest purity available and purchased from Sigma Chemie GmbH (Taufkirchen, Germany). Animals. Groups of 3 male F344 rats (200–250 g; Charles River, Sulzfeld, Germany) were housed in plastic cages under standard animal laboratory conditions (20 ± 2° C; 50 ± 10% relative humidity; 12 h light/dark cycle) with ad libitum access to rat chow (Altromin®, Altromin, Lage/Lippe, Germany) and drinking water. The

Fig. 1 HPLC analysis of non-radiolabelled reference metabolites (upper panel), metabolites of [5-3H]NNK in a sample obtained from 3 h liver perfusion (middle panel) and of [5-3H]NNK and its metabolites in lung perfusion (lower panel)

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A

B

Fig. 2 A Transformation of NNK-N-oxide towards NNK due to incubation with E. coli HPLC analysis of [5-3H]NNK and its metabolites in plasma obtained from 3 h of lung perfusion before (upper panel) and after incubation with E. coli for 24 h. B Selective transformation of NNAL-Glu towards NNAL due to incubation with β-glucuronidase. HPLC analysis of [5-3H]NNK and its metabolites in plasma obtained from 3 h of liver perfusion before (upper panel) and after incubation with glucuronidase for 24 h

dium between 0 and 180 min, erythrocytes and plasma separated by centrifugation and stored at –20° C until analysis. Total radioactivity was measured in aliquots of plasma and erythrocytes after decolourization with H2O2.

animal experiments were officially approved by the Government of Lower Saxony (504.42502/01-34.89) Perfusion of isolated rat organs. The perfusion of intact isolated rat lungs and livers was performed in vitro as described previously (Foth et al. 1984, 1991, 1995). After anaesthesia with urethane (1.25 g/kg body weight) and heparinisation (2000 IE/kg bw) the lungs were ventilated via a tubing inserted into the trachea with 30 × 1.0 ml/min. The lungs were perfused via a cannula inserted into the truncus of the pulmonary arteries at a constant flow rate of 12 ml/min. The efficiency of capillary perfusion in this system was close to the in vivo situation (Foth et al. 1995). The livers were perfused via a cannula inserted into the portal vein. The medium consisted of bovine erythrocytes suspended in Krebs-Ringer bicarbonate buffer (hematocrit 0.14–0.16 in lung perfusion and 0.29–0.31 in liver perfusion) fortified with 1.0 mg/ml glucose and 15 mg/ml bovine serum albumin. [5-3H]NNK was added to the perfusion medium as a short-term infusion over a period of 30 min via perfusor (Braun, Melsungen, Germany) in order to mimic experimental conditions close to smoking habits. The final concentrations of NNK were 35 nM and 150 µM, respectively. Fourteen 1 ml samples were taken from the perfusion me-

HPLC analysis of NNK and its metabolites. NNK and its metabolites were analyzed in samples of plasma and erythrocytes by a modification of the procedure developed by Carmella and Hecht 1985. After precipitation of proteins by TCA, 200–600 µl of the clear supernatant was adjusted to pH 7.4 with 2 M Tris buffer. Samples were chromatographed on a 4 × 250 mm LiChrospher® 60 RP-selectB 5 µm column fitted with a 4 × 4 mm precolumn (Merck, Darmstadt, Germany) by elution with a gradient which was linear from 100% A to 85% A/15% B in 25 min and linear to 70% A/30% B in 5 min (A: 20 mM Tris buffer, pH 7.2; B: acetonitrile) at a flow rate of 0.7 ml/min. Radioactivity was monitored by liquid scintillation counting (Quickszint Flow 302, Zinser Analytic, Frankfurt, Germany) using a radioactivity monitor fitted with a flow cell (LB 506 C-1, Berthold, Wildbad, Germany). Radioactive metabolites were identified by gaschromatography with unlabelled reference compounds detected by UV at 254 nm (Kontron, Ecking, Germany) Typical HPLC runs of a cocktail with the unlabelled reference compounds and two samples obtained from lung and liver perfusion after 3 h, respectively, containing [5-3H]NNK and its metabolites are shown in Fig. 1. The detection limit for a single peak was 740 dpm corresponding to 1% of the total radioactivity in a sample. Enzymatic characterization of NNAL-N-oxide and NNAL-Glu. 1. NNAL-N-oxide (Atawodi and Richter 1996): Aliquots of 1 ml plasma obtained from lung perfusion were incubated with E. coli (HB101, 109 cells) for 24 h at 37° C. Then, plasma and bacteria

339 were separated by centrifugation. Further treatment and quantification of the metabolites were performed as described above (Fig. 2 A). 2. NNAL-Glu (Morse et al. 1990): Aliquots (600 µl) of plasma obtained from liver perfusion were adjusted with HCl (1 N) to pH 6–6.5 and incubated with β-glucuronidase (4 U) for 30 min at 37° C. After incubation, the reaction was stopped by adding trichloroacetic acid. Separation and quantitation of the metabolites were performed as described above. Kinetic parameters and statistics. The concentration versus timecurves were fitted by the TOPFIT V 2.0 calculation programme (provided by the German pharmaceutical companies Gödecke, Schering and Thomae) according to a standard two compartmental model assuming first order kinetics. Pharmacokinetic parameters, such as area under the concentration versus time-curve (AUC), clearance (Cl) and volume of distribution (VD) were derived according to the procedure discribed by Gibaldi and Perrier 1982. AUC was determined by the trapezoidal method and were extrapolated to infinity. Clearance was calculated as dose/AUC. Reported values represent means ± standard error. Statistical analysis was performed by the two-sided Students t-test for independent samples.

Table 1 Pharmakokinetic parameters of NNK elimination in isolated perfused rat lungs and livers 150 µM NNK

35 nM NNK

Cmax (pmol/ml) T1/2 (min) CL (ml/min) VD (ml)

Lung

Liver

Liver

21.8 ± 3.2 47.9 ± 7.4 2.1 ± 0.5 126.0 ± 5.3

14.8 ± 2.9 14.6 ± 1.2 6.9 ± 1.6 143.4 ± 32.4

74.2; 68.0a 35.2; 36.0 2.9; 3.3 153; 169

Rat lungs were perfused with [5-3-H]NNK for 3 h. The volume of perfusion medium was 100 ml, flow rate was set to 12 ml/min, Cmax, maximum plasma concentration; t1/2, elimination half-life; Cl, clearance; VD, volume of distribution Mean ± SE of 5 experiments a Results of two experiments, nmol/ml

Results The metabolism of [5-3H]NNK in rat lung and liver was investigated using the model of isolated perfused organs and, therefore, the intact rat lungs and livers retain their physiological morphology and remain metabolically active for several hours. The experiments were performed using a closed perfusion system, in which the medium repeatedly recirculates through the vessels and capillaries of the intact organ. In order to adapt the experimental conditions the kinetics of substrate load during smoking NNK was administered over a period of 30 min, yielding a final concentration in the medium of 35 nM and 150 µM, respectively. We decided to use a constant rate of input instead of mimicking puffs in order to avoid too complex conditions for kinetic analysis. In all samples analyzed, the bulk of radioactivity was found in the plasma fraction with only 10% of radioactivity remaining in the pelleted erythrocytes. In previous experiments this amount of radioactivity in the pelleted erythrocytes was analyzed in detail. Only NNK could be detected in most erythrocyte fractions. NNK metabolites were not detectable in the erythrocyte fraction before NNK was largely metabolized. In these samples, the qualitative composition of NNK metabolites was identical in the plasma and erythrocyte fractions (data not shown) indicating that NNK and its metabolites are evenly distributed throughout the perfusion medium. Kinetics of NNK elimination in the isolated perfused rat lung and liver In lung as well as in liver the kinetics of NNK can be ascribed by a one-compartment model of elimination. The calculated volume of distribution of NNK was in all cases only slightly larger than the volume of the ‘central’ compartment (Table 1) which is composed of the volume of

Fig. 3 NNK elimination in the isolated rat lungs and livers perfused with 35 nM [5-3H]NNK. Values are the mean ± SE of 5 experiments

perfusion medium plus the intravascular space of the organ. NNK was rapidly removed from the perfusion circuit in both the isolated rat lung and liver, perfused with 35 nM NNK, but NNK elimination in liver was substantially faster than NNK elimination in lung (Fig. 3). Accordingly, the clearance in liver (6.9 ± 1.6 ml/min) was almost 3.3fold higher than the value of pulmonary clearance of 35 nM NNK (2.1 ± 0.5 ml/min) and the half-life of NNK in liver nearly was only 30% of the half-life observed in lung (Table 1). While NNK was completely metabolized in the perfused rat liver within 120 min, about 10.2 ± 2.3% of NNK remained in the perfusion medium of rat lungs after 3 h, indicating a substantially higher metabolic capacity towards NNK in the rat liver compared to the rat lung (Table 2). When liver was perfused in the presence of the high dosage of 150 µmol NNK, which is sufficient to induce liver tumors in F344 rats by repeated subcutaneous injections (Hoffmann et al. 1984), elimination half-life was increased to 36 min, whereas clearance decreased to 3.3 ml/min (Table 1).

340 Table 2 Metabolites of NNK in the medium of isolated perfused rat lung and liver Metabolites

Lung n=5 Hydroxy acid Keto acid Keto alcohol Diol NNAL-N-oxide NNK-N-oxide NNAL-Gluc NNAL NNK

150 µM NNK

35 nM NNK Liver n=3 pmol/ml

Liver n=2

0.1 ± 0.1 3.7 ± 0.7 1.3 ± 0.2 5.2 ± 0.6

16.9 ± 1.7 8.0 ± 1.2 n.d. n.d.

29.1 ± 3.7 30.4 ± 0.6 n.d. n.d.

2.3 ± 0.3 17.1 ± 1.3

3.1 ± 0.5 1.6 ± 0.8

7.0 ± 1.8 3.4 ± 0.5

n.d. 1.8 ± 0.4 3.6 ± 0.8

1.4 ± 0.4 n.d. n.d.

36.7 ± 7.6 2.0 ± 2.0 5.0 ± 2.5

Rat lungs and livers were perfused with [5-3H]NNK for 3 h NNK and its metabolites were determined after 3 h of perfusion Mean ± SE of 3–5 experiments or mean and range of 2 experiments (liver 150 µM) 11% (total dose = 3.5 nmol NNK) or 24% (total dose 15000 nmol NNK) of the total radioactivity was excreted into the bile fluid within 3 h

Fig. 4 N-oxidation of NNK and NNAL in the isolated rat lung and liver perfused with 35 nM [5-3H]NNK. Values are the mean ± SE of 5 experiments. The sum of NNK-N-oxide and NNAL-N-oxide served as a measure for N-oxides. 100% or radioactivity refers to the sum of NNK and metabolites (35 pmol/ml for lung and 31 pmol/ml for liver)

Formation of NNK metabolites Along with the differences in the velocity of NNK disappearance from the perfusion medium the pattern of NNK metabolites obtained after 3 h of perfusion substantially differed between the target organ lung and the liver. When the liver was perfused with 35 nM NNK hydroxy acid, a product of NNK α-hydroxylation, was the main metabolite contributing to 54.3 ± 5.5% to total metabolites. In contrast, in perfused rat lung NNK-N-oxide was quantitatively the main metabolite which indicates that in pulmonary metabolism N-oxidation of NNK towards NNKN-oxide predominates (Table 2). NNK-N-oxide cannot be identified by gas chromatography and mass spectrometry. Also HPLC analysis using diode array detection failed to characterize this metabolite which is supposed to be NNK-N-oxide. In order to validate the identification of the peak which co-elutes with the reference compound NNK-N-oxide to be the main metabolite of NNK in the lung, a sample, taken at 180 min of perfusion was incubated with E. coli (109/ml cells) at 37° C for 24 h prior to HPLC analysis. These bacteria have been demonstrated to selectively reduce the pyridyl N-oxide of NNK-N-oxide and NNAL-N-oxide, yielding NNK and NNAL, respectively (Atawondi and Richter 1996). As shown in Fig. 2 A an incubation of perfusion medium with E. coli quantitatively converted peaks coeluting with NNK-N-oxide and NNAL-N-oxide towards substances co-eluting with NNK and NNAL, respectively, which strongly supports that the peak of interest is NNKN-oxide. The products of detoxification pathways in NNK metabolism, N-oxidation of NNK and NNAL, were predominant in the lung with 55% compared to 15% in the liver (Fig. 4). The sum of metabolites originating from α-hy-

Fig. 5 α-hydroxylation of NNK and NNAL in the isolated rat lung and liver perfused with 35 nM [5-3H]NNK. Values are the mean ± SE of 5 experiments. α-hydroxylation was calculated by the sum of hydroxy acid, keto alcohol and diol. 100% or radioactivity refers to the sum of NNK and metabolites (35 pmol/ml for lung and 31 pmol/ml for liver)

droxylation which is proposed to be the activating metabolic pathway on the one hand, amounted to 80.0% of total metabolites in the liver and to 29.3% of total metabolites in the lung (Fig. 5). In perfused liver only hydroxy acid and keto acid were detectable as metabolites resulting from α-hydroxylation of NNK and NNAL. In contrast, NNK metabolism by lung yielded also amounts of diol and keto alcohol with 15% and 4% after 3 h. Keto acid contributed to 10% of total metabolites and hydroxy acid only to very low amounts in the rat lung (Table 2). As it has to be expected, NNAL-Glu was detected in liver perfusion medium, but this metabolite amounted to 5% of total metabolites (Table 2). However, NNAL-Glu contributed to almost all of the radioactivity excreted into the bile. Altogether, 11% of the administered dose of radioactivity were excreted into the bile. In isolated rat liver perfused with a high concentration of 150 µM NNK the rate of glucuronidation of NNK was

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increased (Table 2). Peak identification of NNAL-Glu was also confirmed using the β-glucuronidase assay as described previously by Morse et al. 1990. Treatment of NNAL-Glu with glucuronidase leads to a selective transformation towards NNAL. As demonstrated in Fig. 2 B the peak co-eluting with NNAL-Glu was quantitatively transformed towards a peak co-eluting with NNAL after 15 min of incubation. Simultaneously to the increased fraction of glucuronides in perfusion medium, the fraction of hydroxy acid was reduced to nearly half of the level obtained at an admininstered dose of 35 nM NNK. The fraction of glucuronidated metabolites was increased by almost the same magnitude as the fraction of metabolites from α-hydroxylation was decreased.

Discussion This study demonstrates that NNK at a concentration of 35 nM is rapidly eliminated in perfused rat livers and also in rat lungs. The kinetics of NNK in isolated livers was substantially faster compared to the kinetics in isolated lung, indicated by a shorter elimination half-life and approximately 3fold higher clearance. However, when the weight of these organs are taken into account the pulmonary clearance (ml/(min × g) of NNK is about 2.4-fold of the hepatic clearance. Altogether, the clearances observed in isolated lung (2.1 ± 0.5 ml/min) and liver (6.9 ± 1.6 ml/min) accounted for about 9 ml/min in vitro which even exceeds the total body clearance in vivo. In F344 rats in vivo NNK is eliminated with a clearance of 5.3 ml/min and a half-life of 25 min (Adams et al. 1985). Therefore, it may be assumed that NNK is cleared from the blood predominantely by lung and liver and that the liver is the major site of NNK metabolism in vivo. NNK clearance is almost exclusively mediated by metabolism and the metabolites are excreted into the urine. Hydroxy acid and keto acid contributed about two thirds of the urinary NNK metabolites in Fisher F344 (F344) rats, which indicates that α-hydroxylation of NNK prevails in vivo and the formation of genotoxic intermediates may occur. The third part of NNK metabolites in the urine of rats is mainly composed of N-oxides of NNK and NNAL. Only very small amounts of NNAL and NNALGlu are excreted into the urine of rats (Kutzer et al. 1995). In humans NNAL-Glu as well as free NNAL have been detected in the urine of smokers, snuff dippers and nonsmokers experimentally exposed to high concentrations of sidestream cigarette smoke (Carmella et al. 1993; Hecht et al. 1993 a; Murphy et al. 1994). The lung is the major target organ in NNK-induced carcinogenesis, this is a consistent finding in a number of studies regardless of the site of NNK application (Hoffmann et al. 1996, for review). However, N-oxidation of NNK, which is suggested to constitute a detoxification pathway, was predominant in the isolated perfused lung, yielding a fraction of 50% of NNK-N-oxide after 3 h perfusion. The identity of NNK-N-oxide was strongly under-

lined by incubating the perfusion medium with E. coli, which selectively transformed NNK-N-oxide and NNALN-oxide towards NNK and NNAL, respectively (Atawodi and Richter 1996). The pathway of α-hydroxylation, which represents metabolic activation of NNK, contributed to 30% in lung. This fraction amounts to less than the half of the fraction observed in the liver. Substantial differences were also observed with regard to the regioselectivity of α-hydroxylation of NNK. In liver perfusions exclusively hydroxy acid as well as keto acid were detected in perfusion medium at any time of sampling. In the isolated perfused lung substantial amounts of diol and keto alcohol resulting from methyl hydroxylation of NNK and NNAL were formed. α-hydroxylation of NNK and NNAL is suggested to take place via two different pathways, which differ in their biological effects (Hecht 1996, for review). Whereas methylene hydroxylation leads to DNA methylating agents, methyl hydroxylation of the two procarcinogenic N-nitrosamines gives rise to DNA pyridyloxobutylation. The formation of O6-mG, which is one of three identified methyl adducts caused by NNK exposure, has been suggested to be crucial in NNK-induced carcinogenesis in A/J mouse (Peterson and Hecht 1991; Hecht et al. 1990; Belinsky et al. 1992). However, other factors have to be important in rat (Van Benthem et al. 1994). An interesting property of pyridyloxobutylated DNA is its ability to inhibit O6-mG-DNA methyltransferase, which leads to a prolonged persistence of O6-mG-DNA (Peterson et al. 1993). Owing to the possible transformation of keto alcohol and diol towards keto acid and hydroxy acid, respectively, the two pathways of α-hydroxylation cannot be discriminated from another with certainty. In our study pyridyloxobutylation was not observed in isolated liver perfusion (detection limit was 1% of the total radioactivity in the plasma sample) but keto alcohol and diol were formed by lung. This is consistent with investigations of Murphy et al. 1990, who revealed a higher level of keto alcohol formation in the lung than in the liver when pyridyloxobutyl DNA adducts were hydrolyzed. This effect was observed in experiments using NNK dosages of less than 150 µg/kg/day. It was more pronounced when the dosage of NNK was decreased. Altogether, these results indicate that DNA pyridyloxobutylation plays a role in NNK-induced carcinogenesis in the rat lung which has already been shown to be important for NNK-induced tumors in rat nasal cavity (Trushin et al. 1994). Hoffmann et al. 1984 demonstrated that NNK is also able to induce liver tumors in F344 rats after chronic exposure towards NNK up to 540 mmol/kg. When a dosage of NNK of 9 mmol/kg was divided into 60 subdoses each of 0.15 mmol/kg (subcutaneous injection) significant numbers of tumors were induced also in liver. Since NNK exhibits a relatively short half-life time (25 min) in vivo (Rivenson et al. 1991, is not substantially bound to proteins, and is a hydrophilic compound, no accumulation of NNK is to be expected within the tissue when NNK is administered repeatedly. The total volume of distribution is

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300 ml for NNK in F344 rats in vivo, and, therefore, a single dose will lead to an initial concentration of approximately 150 µM. When the isolated liver was perfused with 150 µM NNK, a fraction of NNAL-Glu, a metabolite of detoxification, is increased while the fraction of hydroxy acid and keto acid was decreased in parallel when the results were compared with liver perfusions an approximately 4000fold lower dosage of NNK (35 nM). The nature of the peak which co-elutes with NNAL-Glu during HPLC analysis was confirmed by enzymic hydrolysis of NNK metabolites in perfusion medium with β-glucuronidase, yielding a peak co-eluting with NNAL while the peak of NNALGlu was diminished. The increase in NNAL-Glu and the decrease of hydroxy acid reached the same magnitude, indicating that in high dosage the metabolism of NNK in liver was shifted from α-hydroxylation towards NNAL formation. It should be taken in mind, that although detoxification was enhanced an important fraction of NNK still undergoes αhydroxylation, yielding hydroxy acid and keto acid, which still contribute to more than half of the sum of metabolites. Unmetabolized NNK was barely detected in perfusion medium after 3 h. These data demonstrate the high capacity of rat liver to activate NNK also at high dosages where also the liver is a target organ for carcinogenesis due to NNK. In summary, the present study on isolated rat lung and liver shows that the pattern of NNK metabolites is substantially different between lung and liver. In liver α-hydroxylation of NNK prevails and, therefore, it is likely that the genotoxic species of NNK metabolites are formed. The detoxification of NNK by N-oxidation is a prominent metabolic pathway in lung but we obtained strong evidence that pyridyloxobutylating of NNK may be important in lung. In principle, both organs are active with respect to elimination and metabolism of NNK. The clearance of NNK by liver is more than twofold higher than in lung. However, when the clearance is calculated for a gram of organ or for metabolically active cell forms, the capacity in lung to metabolize NNK exceeds that in liver. Acknowledgements We extend special thanks to Dr. D. Hoffmann for providing reference compounds and to G. Rüdell for excellent technical assistance.

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