Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone in patas monkey lung and liver microsomes

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1 Carcinogenesis vol.18 no.8 pp , 1997 Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone in patas monkey lung and liver microsomes Theresa J.Smith 1,3, Anita M.Liao 1, Yuan Liu 1, which was 5-fold higher than human lung and liver microsomes, Ann Butler Jones 2, Lucy M.Anderson 2 and respectively. Immunoblot analysis demonstrated Chung S.Yang 1 that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ and 2 Laboratory of Comparative Carcinogenesis, human liver microsomal sample. Phenethyl isothiocyanate, National Cancer Institute, Frederick, MD 21702, USA an inhibitor of NNK activation in rodents and humans, 3 To whom correspondence should be addressed decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is 50% inhibition of the activity (IC 50 ) values of µm a potent tobacco-specific carcinogen in animals. Our previ- and µm, respectively. The results demonstrate the ous studies indicated that there are differences between similarities and differences between species in the metabolic rodents and humans for the enzymes involved in the activation of NNK. The patas monkey microsomes appear activation of NNK. To determine if the patas monkey is a to more closely resemble human microsomes than mouse better animal model for the activation of NNK in humans, or rat enzymes and may better reflect the activation of we investigated the metabolism of NNK in patas monkey NNK in humans. lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone Introduction (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK*) is a (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1- potent tobacco-specific nitrosamine which induces lung tumors butanol (NNAL) was observed, displaying apparent K m in all laboratory animal species tested (1 3). NNK has also values of 10.3, 5.4, 4.9, and 902 µm, respectively. NNK been shown to induce nasal cavity, pancreatic, and liver tumors metabolism in liver microsomes resulted in the formation in rats (2,3). From studies on the occurrence, carcinogenicity of keto aldehyde, keto alcohol, and NNAL, displaying and metabolic activation of tobacco-specific nitrosamines, apparent K m values of 8.1, 8.2, and 474 µm, respectively. NNK has been suggested to play a role in human tobacco- The low K m values for NNK oxidation in the patas monkey related cancers (2,3). In order for NNK to exert its carcinolung and liver microsomes are different from those in genicity, it must be metabolically activated. Metabolic activahuman lung and liver microsomes showing K m values of tion of NNK involves α-hydroxylation of the methyl and µm, although loss of low K m forms from human methylene carbons of NNK leading to the formation of tissue as a result of disease, surgery or anesthesia cannot be electrophiles which can pyridyloxobutylate and methylate ruled out. Carbon monoxide (90%) significantly inhibited DNA, respectively (2,4). NNK can also undergo pyridine N- NNK metabolism in the patas monkey lung and liver oxidation, and carbonyl reduction. Pyridine N-oxidation of microsomes by 38 66% and 82 91%, respectively. Nordihy- NNK (NNK-N-oxide) is considered to be a detoxification droguaiaretic acid (a lipoxygenase inhibitor) and aspirin pathway, whereas 4-(methylnitrosamino)-1-(3-pyridyl)-1- (a cyclooxygenase inhibitor) decreased the rate of formation butanol (NNAL) (from the carbonyl reduction pathway) has of keto aldehyde and keto alcohol by 10 20% in the monkey been shown to be as carcinogenic as NNK (1,2,4). Pyridine lung microsomes. α-napthoflavone and coumarin markedly N-oxidation and glucuronidation of NNAL have been suggested decreased the oxidation of NNK in monkey lung and liver to be detoxification pathways (4). microsomes, suggesting the involvement of P450s 1A and The various metabolic pathways of NNK have been demon- 2A6. An antibody against human P450 2A6 decreased the strated to occur in vitro and/or in vivo in rodents (1,2,5 8), oxidation of NNK by 12 16% and 22 24% in the patas primates (9,10), and humans (11 16). In mouse and hamster monkey lung and liver microsomes, respectively. These lung microsomes, α-hydroxylation of NNK has been shown results are comparable to that obtained with human lung to be a major pathway (5,7,8). On the other hand, carbonyl and liver microsomes. Coumarin hydroxylation was reduction of NNK is the major metabolic pathway in human lung microsomes and explants (11 13). Recently, two NNAL- glucuronide diastereomers have been detected in the urine of patas monkeys treated with NNK (9), and in urine of smokers and snuff dippers (14 16). In NNK-treated mice and rats, only observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, *Abbreviations: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450, cytochrome P450; NNK N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl- N-oxide)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone; keto aldehyde, 4-oxo-1-(3- pyridyl)-1-butanone; HPLC, high performance liquid chromatography; TAO, troleandomycin; NDGA, nordihydroguaiaretic acid; PEITC, phenethyl isothiocyanate; IC 50, inhibitor concentration resulting in 50% inhibition of the activity. one urinary NNAL-glucuronide diastereomer was detected and it was a minor metabolite at low doses of NNK (17). The extent of NNK metabolism that occurs in the different species may depend on the composition of enzymes present in the tissues. Cytochrome P450 (P450) enzymes have been clearly demon- Oxford University Press 1577

2 T.J.Smith et al. strated to be involved in the activation of NNK in animal and saturated barium hydroxide. Each sample was centrifuged and filtered, and 0.2 ml was coinjected with 5 µl of NNK metabolite standards onto a human tissue microsomes (5,6,11,12,18). P450s 1A2, 2A1, reverse-phase HPLC system equipped with a radioflow detector (Radiomatic and/or 2B1 are responsible for the oxidation of NNK in rat Instruments and Chemical Co., Tampa, FL) (5). The HPLC conditions used and mouse lung microsomes (6,7). In rat liver microsomes, were the same as previously described (18). P450s 1A2, 2A1, and 3A account for only a fraction of the For kinetic studies, NNK concentrations of 1, 5, 10, 20, 50, 100, 200, 500, activity of NNK oxidation (18). Our studies have suggested and 1000 µm were used in incubations. For inhibition studies, air or a mixture of carbon monoxide and air (9:1) was bubbled through the microsome-buffer that P450 2A6 or a P450 2A6-related enzyme is involved in mixture for 3 min before using in incubations or the chemical inhibitors were the activation of NNK in human lung microsomes (12). In dissolved in methanol and used at 0.5% of the total incubation volume. At human liver microsomes, P450s 1A2, 2A6, 2E1, and 3A4 have this concentration, methanol had no effect on NNK metabolism. When TAO been shown to be responsible for NNK activation (11,19). was used, the microsomes were preincubated with 1 mm NADPH and 50 µm Using expressed human P450s, it has been demonstrated that TAO for 30 min at 37 C. The reaction was then initiated with NNK and 1 mm NADPH. P450s 1A2, 2A6, 2B7, 2D6, 2E1, 2F1, 3A4, and 3A5 catalyzed Coumarin metabolism analysis the α-hydroxylation of NNK (11,19,20). Of these expressed Coumarin 7-hydroxylation was determined as described (25). In brief, incuba- P450s, P450s 1A2 and 2A6 showed the lowest K m values tions contained 50 mm Tris HCl (ph 7.4), 1 mm NADPH, 50 µm coumarin, ( µm) in the formation of 4-oxo-1-(3-pyridyl)-1- and 0.05 mg (liver) or 0.2 mg (lung) microsomal protein in a total volume of butanone (keto aldehyde) and 4-hydroxy-1-(3-pyridyl)-1-but- 0.5 ml. Reactions were carried out for 15 min at 37 C. Samples were extracted, anone (keto alcohol) (α-hydroxylation products) (11,19). In centrifuged and the formation of 7-hydroxycoumarin was measured by human lung microsomes, the P450-dependent pathways only fluorescence with λ excitation of 358 nm and λ emission of 458 nm. The enzyme activities were calculated by comparison with a standard curve of 7-hydroxyaccount for 5 49% of the activities for NNK oxidation (12), coumarin which was subjected to the same incubation and extraction procedures whereas P450s are fully responsible for NNK activation in as the samples. rodent lung (5,6). It has recently been demonstrated that Immunoblot analysis lipoxygenase and reactive oxygen species, particularly Liver microsomal proteins were separated by sodium dodecyl sulfate-polyacrylhydroxyl radicals and superoxide anion, may play a role in amide gel electrophoresis and transferred to a nitrocellulose sheet. Antibodies the bioactivation of NNK (12,21,22). which recognize P450s 1A1/2 and 2A6 were used and the blots were It is difficult to extrapolate results for NNK metabolism subsequently immunostained by the ECL TM western blotting system (Amersham, Arlington Heights, IL). Densitometry was performed using a Bio from the rodent to humans due to species differences in P450s. Image Intelligent Quantifier/Omni Scanner. Since primates are closely related to humans, the present study Data analysis investigated the metabolism of NNK in patas monkey lung The apparent kinetic parameters were determined by curve-fitting the data and liver microsomes and characterized the enzymes involved to the Michaelis Menten equation using KaleidaGraph (Synergy Software, in the activation. The results obtained with the patas monkey Reading, PA), a non-linear regression data analysis program. Data were were compared with our previous results in humans and rodents. statistically analyzed by the Student s t-test or by analysis of variance. Materials and methods Results Chemicals NNK metabolism Unlabeled NNK and [5-3 H]NNK (2.4 Ci/mmol; purity 97%) were purchased In the patas monkey lung microsomes, the formation of from Chemsyn Science Laboratories (Lenexa, KS). The radiolabeled NNK keto aldehyde, NNK-N-oxide, keto alcohol and NNAL was was further purified by reverse-phase high performance liquid chromatography observed, with keto alcohol being the major metabolite (Figure (HPLC) before use. Authentic NNK metabolite standards were kindly provided 1). The rate of the formation of keto alcohol was 6.8- by Dr Stephen Hecht (University of Minnesota Cancer Center, Minneapolis, MN). NADPH, NADP, glucose 6-phosphate, glucose-6-phosphate dehydromicrosomes, the formation of keto aldehyde, keto alcohol and fold greater than keto aldehyde. In the patas monkey liver genase, coumarin, troleandomycin (TAO), α-napthoflavone, aspirin, and nordihydroguaiaretic acid (NDGA) were purchased from Sigma Chemical Co. NNAL was observed, with NNAL being the major metabolite (St Louis, MO). Phenethyl isothiocyanate (PEITC) and 7-hydroxycoumarin (Figure 1). The formation of keto aldehyde and keto alcohol were from Aldrich Chemical Co (Milwaukee, WI). The monoclonal antibody against human P450 2A6 was purchased from Gentest Corp (Woburn, MA). occurred at a similar rate. Total NNK metabolism was 3-fold The polyclonal antibody against rat P450 1A1/2 was generously supplied by greater with the liver microsomes than the lung microsomes Dr Paul Thomas (Rutgers University, Piscataway, NJ). All other chemicals due to the extensive formation of NNAL in the liver microwere of reagent grade. somes. The addition of 5 mm sodium bisulfite (for trapping Monkey lung and liver microsomes keto aldehyde) had no inhibitory effect on the metabolism of A colony-reared female patas monkey (No. 665) was maintained and treated NNK in monkey lung and liver microsomes and the formation under AAALAC-approved conditions at BioQual, Inc. (Rockville, MD), in of the NNK metabolites was linear with time and protein accordance with humane principles for laboratory animal care. Protocols were concentration (data not shown). reviewed and approved by the BioQual, Inc. Animal Care and Use Committee. The monkey was sedated with a high dose of ketamine and exsanguinated. Kinetic parameters Organs were rapidly removed and immediately chilled prior to preparation of For lung and liver microsomes, 7 and 9 concentrations of microsomes by differential centrifugation followed by washing and resuspension in 0.25 M sucrose (23). Microsomes were stored at 80 C. The protein NNK ( µm) were used, respectively, to determine the concentration was determined according to Lowry et al. (24), using bovine kinetic parameters in the activation of NNK. Michaelis Menten serum albumin as the standard. kinetics were observed in the formation of keto aldehyde, NNK metabolism analysis NNK-N-oxide, and keto alcohol in the patas monkey lung and Unless otherwise stated, incubations consisted of 100 mm sodium phosphate liver microsomes when NNK concentration ranges of 1 20 (ph 7.4), 1 mm EDTA, 3 mm MgCl 2, an NADPH-generating system (5 mm µm (lung) and 1 50 µm (liver) were used (Figure 2A and B). glucose 6-phosphate, 1 mm NADP, and 1.5 units glucose 6-phosphate The apparent K m and V max values are summarized in Table I. dehydrogenase), 10 µm NNK (containing 1 µci [5-3 H]NNK), 5 mm sodium bisulfite, and 0.1 mg (liver) or 0.2 mg (lung) microsomal protein in a total In the patas monkey lung microsomes, the apparent K m for volume of 0.4 ml. Reactions were carried out for 20 min (liver) or 30 min the formation of keto aldehyde (10.3 µm) was 2-fold higher (lung) at 37 C and terminated with 100 µl each of 25% zinc sulfate and than the K m for keto alcohol (4.9 µm). The apparent K m for 1578

3 NNK metabolism in monkey lung and liver microsomes Fig. 1. NNK metabolism in patas monkey lung and liver microsomes. Incubations contained 10 µm NNK (containing 1 µci [5-3 H]NNK), an NADPH-generating system, 5 mm sodium bisulfite and 0.2 mg (lung) or 0.1 mg (liver) microsomal protein. Reactions were carried out for 30 min (lung) or 20 min (liver) at 37 C. Values are the mean SD (bars) of two replicates. Fig. 2. Substrate dependency for the oxidation of NNK by patas monkey lung (A) and liver (B) microsomes. Monkey lung or liver microsomes were incubated with 1 20 µm (lung) or 1 50 µm (liver) NNK as described in Materials and methods. Reactions were carried out for 30 min (lung) or 20 min (liver) at 37 C and the formation of keto aldehyde (j), NNK-N-oxide (r) and keto alcohol (d) was determined. Points are the mean of two replicates; the difference between replicates was 10%. the formation of NNK-N-oxide (5.4 µm) was similar to the K m for keto alcohol. The formation of NNAL, possibly due to the activity of carbonyl reductase, however, displayed a very large K m (902 µm). In the patas monkey liver microsomes, the apparent K m (8.1 µm) and V max (37 pmol/min/mg protein) values were the same in the formation of keto aldehyde and keto alcohol, suggesting that the same enzyme may be involved in the formation of both metabolites. In addition to the low K m enzymes, other enzymes are involved in the formation of keto aldehyde, NNK-N-oxide and keto alcohol in the patas monkey lung and liver microsomes because saturation kinetics for the formation of these metabolites were not observed in the NNK concentration range of µm (data not shown). Effect of inhibitors on NNK metabolism In order to determine the contributions of P450, cyclooxygenase, lipoxygenase, and flavin-containing monooxygen- ase to the metabolism of NNK, inhibitors of these enzymes were used (Tables II and III). In the patas monkey lung microsomes, the rate of formation of keto aldehyde, NNK-Noxide, keto alcohol, and NNAL was decreased by carbon monoxide by 48, 61, 66, and 38%, respectively. Aspirin, a cyclooxygenase inhibitor, decreased keto alcohol formation by 10%. NDGA, a lipoxygenase inhibitor, decreased the formation of keto aldehyde and keto alcohol by 20 and 15%, respectively. Preincubation of the microsomes in the absence of NADPH, which was intended for the inactivation of the flavin-containing monooxygenase, decreased the formation of NNK-N-oxide and keto alcohol by 12 and 8%, respectively (Table II). In the patas monkey liver microsomes, carbon monoxide decreased the rate of formation of keto aldehyde, keto alcohol, and NNAL by 89, 91, and 82%, respectively. Aspirin, NDGA, and preincubation had no significant effects on NNK metabolism in monkey liver microsomes (Table III). The results suggest that P450 plays a major role in the metabolism of NNK in the patas monkey liver microsomes, whereas in the patas monkey lung microsomes, P450 is only partially involved in NNK metabolism. To determine the P450 forms that are involved in the activation of NNK in the patas monkey lung and liver microsomes, various chemical inhibitors were used. α-napthoflavone, coumarin and TAO are selective inhibitors of P450s 1A, 2A6, and 3A in human liver microsomes (26 28), respectively. In monkey lung microsomes, α-napthoflavone and coumarin decreased the oxidation of NNK by 11 33% and 10 40%, respectively (Figure 3). In monkey liver microsomes, α-napthoflavone significantly inhibited the formation of keto alcohol by 43%. Coumarin decreased the rate of formation of keto aldehyde and keto alcohol by ~60% (Figure 3). TAO had no appreciable effects on NNK metabolism in monkey lung and liver microsomes. The results suggest that P450s 1A and 2A6 may be involved in the oxidation of NNK in the patas monkey lung and liver microsomes. To investigate the involvement of P450 2A6 in the oxidation of NNK in the patas monkey lung and liver microsomes, a monoclonal antibody against human P450 2A6 was used. In lung microsomes, the rate of formation of keto aldehyde and keto alcohol was decreased by 12% and 16%, respectively. The antibody had no effect on NNK-N-oxide and NNAL formation. In liver microsomes, the formation of keto aldehyde and keto alcohol was decreased by 22% and 24%, respectively (Table IV). 1579

4 T.J.Smith et al. Table I. Kinetic parameters for NNK metabolism in patas monkey lung and liver microsomes a Metabolites K m (µm) V max (pmol/min/mg protein) Lung Liver Lung Liver Keto aldehyde NNK-N-oxide ND b ND Keto alcohol NNAL a The kinetic parameters for keto aldehyde, NNK-N-oxide, and keto alcohol formation in patas monkey lung and liver microsomes were determined using four and five concentrations of NNK, respectively. For NNAL formation, seven and nine concentrations of NNK were used in patas monkey lung and liver microsomes, respectively. Values are the mean SD of two replicates. b ND, metabolite was not detectable. Table II. Inhibition of NNK metabolism in monkey lung microsomes by various inhibitors a Inhibitor conc. (µm) Keto aldehyde NNK-N-oxide Keto alcohol NNAL % inhibition Carbon monoxide b 90% Aspirin Nordihydroguaiaretic acid Preincubation c a Control lung microsome values were 3.0, 6.8, 11.7 and 5.2 pmol/min/mg protein for keto aldehyde, NNK-N-oxide, keto alcohol, and NNAL, respectively. Values are the mean SD of three determinations. Values in the same group with different superscripts are significantly (P 0.05) different from the control and each other. b A mixture of carbon monoxide and air (90:10%) was bubbled through the microsome-buffer mixture for 3 min before using in incubations. c The microsome-buffer mixture was preincubated at 37 C for 10 min in the absence of NADPH. Table V. In the individual patas monkey liver microsomal sample, Table III. Inhibition of NNK metabolism in monkey liver microsomes by bands of similar molecular weights to those of human liver P450s various inhibitors a 1A and 2A6 were observed. In comparison to an individual Inhibitor conc. (µm) Keto aldehyde Keto alcohol NNAL human liver microsomal sample (HL 6 from the HepatoScreen TM test kit, Human Biologics, Inc., Phoenix, AZ), the P450 2A level % inhibition in the individual patas monkey liver microsomal sample was ~6- Carbon monoxide b fold higher. Immunoblotting experiments could not detect P450s 90% A and 2A in the patas monkey lung microsomal sample even Aspirin when 100 µg of protein was used Coumarin metabolism Nordihydroguaiaretic acid In order to examine further the presence of P450 2A in the patas monkey lung and liver microsomes, the metabolism of coumarin Preincubation c was determined. In human liver microsomes, coumarin hydroxylation is a P450 2A6-specific reaction (29,30). Monkey lung and liver microsomes catalyzed the formation of 7-hydroxya Control liver microsome values were 20.9, 21.6 and 62.9 pmol/min/mg coumarin from coumarin (50 µm) at a rate of 16 and 4000 pmol/ protein for keto aldehyde, keto alcohol and NNAL, respectively. Values are min/mg protein, respectively. The results suggest that P450 2A6 the mean SD of three determinations. Values in the same group with different superscripts are significantly (P 0.05) different from the control or a related enzyme is present in the patas monkey lung and liver and each other. microsomes. A mixture of carbon monoxide and air (90:10%) was bubbled through the To investigate further the involvement of P450 2A in the microsome-buffer mixture for 3 min. metabolism of NNK in the patas monkey lung and liver microc The microsome-buffer mixture was preincubated at 37 C for 10 min in the somes, NNK was used as an inhibitor of coumarin metabolism. absence of NADPH. As the NNK concentration increased, the formation of 7-hydroxycoumarin decreased (Figure 4). However, the percent decrease Hepatic microsomal P450s 1A and 2A6 in 7-hydroxycoumarin formation by NNK was less (by 50%) Since the above results suggest that P450s 1A and 2A6 are than that observed when coumarin was used as an inhibitor of involved in the oxidation of NNK, P450s 1A and 2A in the liver NNK metabolism in the lung and liver microsomes (Figure 3). microsomes of the patas monkey were determined by immuno- These results suggest that coumarin may be oxidized by other blot analysis using anti-rat P450 1A and anti-human P450 2A6 P450(s), in addition to P450 2A6, in the patas monkey lung and antibodies. The intensities of the protein bands are shown in liver microsomes. 1580

5 NNK metabolism in monkey lung and liver microsomes Table IV. Inhibition of NNK metabolism in the patas monkey lung and liver microsomes by a monoclonal antibody against human P450 2A6 a Sample Keto aldehyde NNK-N-oxide Keto alcohol NNAL pmol/min/mg protein Lung Control Anti-2A (12) (0) (16) (0) Liver Control 20.6 ND b Anti-2A ND (22) (24) (0) a The antibody against human P450 2A6 was added to lung and liver microsomes at a concentration of 0.1 µg/µg microsomal protein. The microsomeantibody mixture was kept at room temperature for 15 min before using in incubations. Incubations contained 10 µm NNK, an NADPH-generating system, 0.1 mg (liver) or 0.2 mg (lung) microsomal protein, 5 mm sodium bisulfite and 10 µg or20µg anti-2a6. Reactions were carried out at 37 C for 20 min (liver) or 30 min (lung). Values are the average of duplicates. Numbers in parentheses are the percent inhibition. b ND, metabolite was not detectable. Table V. Levels of P450s 1A and 2A6 in the patas monkey and human liver microsomes a Species P450 1A P450 2A6 arbitrary units Monkey Human b a Immunoblot analysis of anti-rat P450 1A and anti-human P450 2A6 was performed with the patas monkey and human liver microsomes. The antibody preparation against P450 1A recognizes P450s 1A1 and 1A2. b The individual human liver microsomal sample was sample HL 6 from the HepatoScreen TM test kit (Human Biologics, Inc., Pheonix AZ). Inhibition of NNK metabolism by PEITC PEITC, a compound derived from cruciferous vegetables, has been shown to be an effective inhibitor of NNK metabolism in rodent lung microsomes and human lung and liver microsomes by inhibiting P450s (5,7,12,31). In the patas monkey lung and liver microsomal incubations, the inhibition of NNK oxidation by PEITC was concentration-dependent (Figure 5). In the patas monkey lung microsomes, the estimated IC 50 values for keto aldehyde, NNK-N-oxide, and keto alcohol formation were 0.80, 0.30, and 0.28 µm, respectively. In liver microsomes, the estimated IC 50 values for keto aldehyde and keto alcohol formation were 6.8 and 4.2 µm, respectively. The formation of NNAL by the lung and liver microsomes was not affected by PEITC at all concentrations. Fig. 3. Effect of selective P450 inhibitors on NNK metabolism in patas Discussion monkey lung and liver microsomes. Incubation conditions are as described At a 10 µm NNK concentration, the patas monkey lung in Materials and methods. Values are the mean SD (bars) of three determinations. For TAO in the monkey lung microsomes, the values are microsomes exhibited high activities for the oxidation of NNK, the average of two determinations. Values with * indicates significantly which is consistent with results obtained with mouse lung (P 0.05) different from the control group as determined by the Student s microsomes (5,7). In contrast, carbonyl reduction of NNK t-test. (NNAL formation) has been observed to be the major pathway in human lung microsomes (11,12) and cultured human lung hydroxylation rate for NNK was greater with the monkey lung explants (13). Furthermore, the formation of keto alcohol was and liver microsomes than human lung and liver microsomes. observed in the patas monkey (Figure 1) and rodent lung This quantitative difference may reflect the level of P450s microsomes (6,7), but not in human lung microsomes (11,12). which are present in the microsomes. These differences in the metabolic profile for human and patas The presence of low K m forms of enzymes for NNK monkey lung microsomes may be due to differences in the oxidation were observed in the patas monkey lung and liver amount of P450s or in the regiospecificity of metabolism by microsomes (Table I and Figure 2). This result is in contrast these enzymes. The metabolic profile for NNK in patas monkey, to the high K m values ( µm) observed in human lung human and rodent liver microsomes are similar in that NNAL and liver microsomes (11,19). Of the expressed human P450s is the major metabolite formed (11,18). However, the α- studied, P450 2A6 exhibited the lowest K m value (118 µm) 1581

6 T.J.Smith et al. Fig. 5. Concentration-dependent inhibition of NNK metabolism in patas monkey lung and liver microsomes by PEITC. Incubations contained 10 µm NNK, NADPH-generating system, 5 mm sodium bisulfite, 0.1 mg (liver) or Fig. 4. Inhibition of coumarin metabolism by NNK in patas monkey lung 0.2 mg (lung) microsomal protein and 0 10 µm PEITC. Reactions were and liver microsomes. Incubations contained 50 µm coumarin, 1 mm carried out for 30 min (lung) or 20 min (liver) at 37 C and the formation of NADPH, 0.05 mg (liver) or 0.2 mg (lung) microsomal protein, and keto aldehyde (j), NNK-N-oxide (m), keto alcohol (d) and NNAL (u) µm NNK. Reactions were carried out for 15 min at 37 C and the formation were determined. Each point is the mean SD of three determinations. of 7-hydroxycoumarin was determined. Rates for the lung microsomes are expressed as pmol/min/mg protein and the rates for the liver microsomes are expressed as nmol/min/mg protein. Each point is the mean SD of and liver microsomes by 38 and 82%, respectively (Tables II three determinations. and III). A general P450 inhibitor, metyrapone (100 µm), also decreased NNAL formation in the monkey lung and liver (19). It is possible that other enzymes are important in the low microsomes by 43 and 83%, respectively (data not shown). K m pathway for NNK oxidation in humans. Carbon monoxide has also been demonstrated to inhibit the P450 enzymes are known to be responsible for the metabolism rate of NNAL formation in human lung microsomes (by 5 of many carcinogens. Similar to the results obtained with 38%), depending on the particular sample examined (12). Our human and rat liver microsomes (11,18), P450s are the major results suggest that P450 may be involved in the formation of enzymes involved in the metabolism of NNK in patas monkey NNAL in the patas monkey and humans. It is also possible liver microsomes (Table III). In contrast, P450s were only that carbon monoxide may be inhibiting other enzymes in partially involved in NNK metabolism in the patas monkey lung addition to P450. Further studies are needed to establish the microsomes (Table II), analagous to human lung microsomes enzymes that catalyze NNAL formation in these species. (11,12). The use of chemical inhibitors suggests that lipoxygen- Previous studies with human lung and liver microsomes ase and cyclooxygenase also play a role in the activation of suggested that the activation of NNK is catalyzed by P450s NNK in the patas monkey lung microsomes. Similar results 1A2, 2A6, 2E1 and/or 3A4 (11,12,19). P450s 1A and 2A6 have been observed with human lung microsomes (12), but appeared to be involved in the oxidation of NNK in the patas not rodent lung microsomes (5,6). In liver microsomes, P450s monkey lung and liver microsomes based on the inhibitory exist at rather high levels and will apparently have a predominant action of α-napthoflavone, coumarin, and an anti-2a6 antibody role in NNK metabolism in the species studied. On the on NNK metabolism (Figure 3 and Table IV), and the inhibition other hand, in patas monkey and human lung microsomes, the of coumarin metabolism (a representative activity for P450 levels of P450s are low and other enzymes such as lipoxygenase 2A6) by NNK (Figure 4). α-napthoflavone is an inhibitor of and cyclooxygenase may also contribute to the activation P450s 1A1 and 1A2 (26). It has been demonstrated that P450 of NNK. 1A2, but not P450 1A1 is present in the patas monkey liver It has been postulated that carbonyl reductase is responsible microsomes (32). Therefore, α-napthoflavone is most likely for the formation of NNAL (1). Surprisingly, carbon monoxide inhibiting P450 1A2 in the patas monkey liver microsomes. decreased the formation of NNAL in the patas monkey lung Studies have shown that P450 1A2 is responsible for the 1582

7 NNK metabolism in monkey lung and liver microsomes formation of keto alcohol in human liver microsomes (11,31). more, the involvement of P450s 1A and 2A in the activation The inhibition of only keto alcohol formation by α-napthoflavone of NNK in the patas monkey lung and liver microsomes is in the patas monkey liver microsomes is consistent similar to that in humans. with the involvement of P450 1A2 in the formation of this metabolite. Immunoblotting experiments demonstrated that the Acknowledgement P450 2A level was ~6-fold greater in the patas monkey This study was supported by NIH grant CA46535 and NIEHS Center liver microsomal sample than in an individual human liver Grant ES microsomal sample (Table V). This result for P450 2A is similar to our coumarin metabolism results in which the References activity was 5-fold higher in the patas monkey liver microsomal sample than in the same individual human liver microsomal 1. Hecht,S.S., Castonguay,A., Rivenson,A., Mu,B. and Hoffmann,D. (1983) Tobacco specific nitrosamines: carcinogenicity, metabolism, and possible sample (data not shown). This higher content for P450 2A role in human cancer. J. Environ. Sci. Health, C1, may account for the greater rate of activation of NNK in the 2. Hecht,S.S. (1994) Metabolic activation and detoxification of tobaccospecific liver microsomes from the patas monkey as compared to nitrosamines a model for cancer prevention strategies. Drug the human. Metab. Rev., 26, Hoffmann,D. and Hecht,S.S. (1985) Nicotine-derived N-nitrosamines and The P450s that are present in patas monkey lung are not tobacco-related cancer: current status and future directions. Cancer Res., known. The monkey lung microsomes displayed low coumarin 45, hydroxylase activity, suggesting that P450 2A6 or a related 4. Hecht,S.S. (1996) Recent studies on mechanisms of bioactivation and enzyme is present in the lung microsomes. The rate for detoxification of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. CRC Crit. Rev. Toxicol., 26, coumarin hydroxylase in the patas monkey lung microsomes 5. Smith,T.J., Guo,Z.-Y., Thomas,P.E., Chung,F.-L., Morse,M.A., Eklind,K. (16 pmol/min/mg protein) was ~5-fold higher than human and Yang,C.S. (1990) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)- lung microsomes (12). Further studies are needed for the 1-butanone in mouse lung microsomes and its inhibition by isothiocyanates. characterization of the P450s present in the patas monkey lung. Cancer Res., 50, PEITC, a chemopreventive agent present in cruciferous 6. Smith,T.J., Guo,Z., Hong,J.-Y., Ning,S.M., Thomas,P.E. and Yang,C.S. (1992) Kinetics and enzyme involvement in the metabolism of 4- vegetables, has been demonstrated to inhibit NNK-induced lung (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in microsomes of tumorigenesis, DNA adduct formation, and NNK oxidation rat lung and nasal mucosa. Carcinogenesis, 13, (5,7,23,31,33 36). The inhibitory activity of PEITC is due to 7. Smith,T.J., Guo,Z., Li,C., Ning,S.M., Thomas,P.E. and Yang,C.S. (1993) blocking the activation of NNK by chemical inactivation and Mechanisms of inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1- competitive inhibition of the enzyme(s) involved (5,7,31,33). butanone (NNK) bioactivation in mouse by dietary phenethyl isothiocyanate. Cancer Res., 53, In the present study, PEITC inhibited the oxidation of NNK 8. Jorquera,R., Castonguay,A. and Schuller,H.M. (1993) Effect of tobacco in vitro, but not the carbonyl reduction of NNK, in the patas smoke condensate on the metabolism of 4-(methylnitrosamino)-1-(3- monkey lung and liver microsomes (Figure 5). These results pyridyl)-1-butanone by adult and fetal hamster microsomes. Drug. Metab. are consistent with our previous studies in rodent lung micro- Dispos., 21, Hecht,S.S., Trushin,N., Reid-Quinn,C.A., Burak,E.S., Jones,A.B., somes, and human lung and liver microsomes Southers,J.L., Gombar,C.T., Carmella,S.G., Anderson,L.M. and Rice,J.M. (5,7,12,23,31,33). Recent studies have shown that when PEITC (1993) Metabolism of the tobacco-specific nitrosamine 4- was given to NNK-treated rats and NNK-treated patas monpharmacokinetics (methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: keys, increased levels of NNAL and NNAL-glucuronide were and characterization of glucuronide metabolites. Carcinogenesis, 14, excreted in the urine due to PEITC blocking the activation of 10. Castonguay,A., Tjalve,H., Trushin,N., d Argy,R. and Sperber,G. (1985) NNK (36 and Stephen Hecht, personal communication). PEITC Metabolism and tissue distribution of tobacco-specific N-nitrosamines in appears to be a potent inhibitor of the patas monkey lung the marmoset monkey (Callithrix jacchus). Carcinogenesis, 6, enzymes showing IC 50 values of µm for NNK 11. Smith,T.J., Guo,Z., Gonzalez,F.J., Guengerich,F.P., Stoner,G.D. and oxidation. This is similar to rodent lung microsomes Yang,C.S. (1992) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone in human lung and liver microsomes and cytochromes P-450 (5,7,23,33), but lower than human lung microsomes which expressed in hepatoma cells. Cancer Res., 52, displayed IC 50 values of 5 µm (12,31). PEITC exhibited 12. Smith,T.J., Stoner,G.D. and Yang,C.S. (1995) Activation of 4- high IC 50 values of µm in the patas monkey liver (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung microsomes, similar to human liver microsomes (31). PEITC microsomes by cytochromes P450, lipoxygenase, and hydroperoxides. Cancer Res., 55, is known to be a potent competitive inhibitor of P450 1A2 13. Castonguay,A., Stoner,G.D., Schut,H.A. and Hecht,S.S. (1983) Metabolism (31), and is expected to inhibit the activation of NNK in the of tobacco-specific N-nitrosamines by cultured human tissues. Proc. Natl patas monkey liver microsomes. Acad. Sci. USA, 80, The present study demonstrates the similarities and differtobacco-specific 14. Carmella,S.G., Akerkar,S. and Hecht,S.S. (1993) Metabolites of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- ences for NNK metabolism betweeen the patas monkey, rodents butanone in smokers urine. Cancer Res., 53, and humans. The major differences between the patas monkey 15. Hecht,S.S., Carmella,S.G., Murphy,S.E., Akerkar,S., Brunnemann,K.D. and human for NNK metabolism were the presence of low K m and Hoffmann,D. (1993) A tobacco-specific lung carcinogen in the urine enzymes and higher activities for the total α-hydroxylation of of men exposed to cigarette smoke. N. Engl. J. Med., 329, NNK in the patas monkey liver and lung microsomes. There 16. Murphy,S.E., Carmella,S.G., Idris,A.M. and Hoffmann,D. (1994) Uptake is a possibility that these differences are due in part to diseaseby sudanese snuff dippers. Cancer Epidem. Biomark. Prev., 3, and metabolism of carcinogenic levels of tobacco-specific nitrosamines or treatment-related decreases in certain P450s in the human 17. Morse,M.A., Eklind,K.I., Toussaint,M., Amin,S.G. and Chung,F.-L. (1990) surgical samples utilized, including suicide inhibition of low Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-1- K m forms by anesthesia. On the other hand, our results in the (3-pyridyl)-1-butanone (NNK) and its dose-dependent excretion in the patas monkey lung microsomes are similar to human lung urine of mice and rats. Carcinogenesis, 11, Guo,Z., Smith,T.J., Thomas,P.E. and Yang,C.S. (1992) Metabolism of 4- microsomes in that P450 enzymes are only partially responsible (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by inducible and for the activation of NNK, and lipoxygenase and cyclo- constitutive cytochrome P450 enzymes in rats. Arch. Biochem. Biophys., oxygenase may play a role in the activation process. Further- 298,

8 T.J.Smith et al. 19. Patten,C.J., Smith,T.J., Murphy,S.E., Wang,M.-H., Lee,J., Tynes,R.E., Koch,P. and Yang,C.S. (1996) Kinetic analysis of activation of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450 specific chemical inhibitors on this activation in human liver microsomes. Arch. Biochem. Biophys., 333, Crespi,C.L., Penman,B.W., Gelboin,H.V. and Gonzalez,F.J. (1991) A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)- 1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6. Carcinogenesis, 12, Weitberg,A. and Corvese,D. (1993) Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines. Clin. Genet., 43, Kim,P.M. and Wells,P.G. (1996) Genoprotection by UDP-glucuronosyltransferases in peroxidase-dependent, reactive oxygen species-mediated micronucleus initiation by the carcinogens 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone and benzo[a]pyrene. Cancer Res., 56, Guo,Z., Smith,T.J., Wang,E., Eklind,K.I., Chung,F.-L. and Yang,C.S. (1993) Structure-activity relationships of arylalkyl isothiocyanates for the inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and the modulation of xenobiotic-metabolizing enzymes in rats and mice. Carcinogenesis, 14, Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, Yun,C.-H., Shimada,T. and Guengerich,F.P. (1991) Purification and characterization of human liver microsomal cytochrome P-450 2A6. Mol. Pharmacol., 40, Halpert,J.R., Guengerich,F.P., Bend,J.R. and Correia,M.A. (1994) Contemporary issues in toxicology. Selective inhibitors of cytochromes P450. Toxicol. Appl. Pharmacol., 125, Chang,T.K.H., Gonzalez,F.J. and Waxman,D.J. (1994) Evaluation of triacetyloleandomycin, α-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochrome P450. Arch. Biochem. Biophys., 311, Waxman,D.J., Lapenson,D.P., Aoyama,T., Gelboin,H.V., Gonzalez,F.J. and Korzekwa,K. (1991) Steroid hormone hydroxylase specificities of eleven cdna-expressed human cytochrome P450s. Arch. Biochem. Biophys., 290, Yamano,S., Tatsuno,J. and Gonzalez,F.J. (1990) The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochem., 29, Miles,J.S., McLaren,A.W., Forrester,L.M., Glancey,M.J., Lang,M.A. and Wolf,C.R. (1990) Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity. Biochem. J., 267, Smith,T.J., Guo,Z., Guengerich,F.P. and Yang,C.S. (1996) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl isothiocyanate. Carcinogenesis, 17, Sadrieh,N. and Snyderwine,E.G. (1995) Cytochromes P450 in cynomolgus monkeys mutagenically activate 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) but not 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Carcinogenesis, 16, Guo,Z., Smith,T.J., Wang,E., Sadrieh,N., Ma,Q., Thomas,P.E. and Yang,C.S. (1992) Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats. Carcinogenesis, 13, Morse,M.A., Amin,S.G., Hecht,S.S. and Chung,F.-L. (1989) Effects of aromatic isothiocyanates on tumorigenicity, O 6 -methylguanine formation and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone in A/J mouse lung. Cancer Res., 49, Morse,M.A., Wang,C.-X., Stoner,G.D., Mandal,S., Conran,P.B., Amin,S.G., Hecht,S.S. and Chung,F.-L. (1989) Inhibition of 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate. Cancer Res., 49, Hecht,S.S., Trushin,N., Rigotty,J., Carmella,S.G., Borukhova,A., Akerkar,S. and Rivenson,A. (1996) Complete inhibition of 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone-induced rat lung tumorigenesis and favorable modification of biomarkers by phenethyl isothiocyanate. Cancer Epidemiol. Biomark. Prev., 5, Received on February 25, 1997; revised on May 6, 1997; accepted on May 12,