Identification of the human liver microsomal cytochrome P450s involved in the metabolism of N-nitrosodi-n-propylamine

John F. Teiber1,2 and Paul F. Hollenberg2,3

1 Department of Environmental and Industrial Health and
2 Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-0632, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability of human liver cytochrome P450s to metabolize the environmental carcinogen N-nitrosodi-n-propylamine (NDPA) was investigated. The maximum rate of NDPA depropylation in seven human liver microsomal samples was 1.15 nmol/min/mg (range 0.53–2.60). Troleandomycin, a P450 3A4/5 inhibitor, inhibited depropylation modestly (10–60%) in three of seven samples. Diethyldithiocarbamic acid, a potent 2E1 inhibitor, and a 2E1 inhibitory monoclonal antibody (mAb) inhibited the reaction in all samples (23 to almost 100%). No significant inhibition was observed with the 2C9 inhibitor sulfaphenazole or with mAbs to 3A4, 2A6 and 2D6. The 2C8/9/18/19 mAb inhibited depropylation in one sample by ~25% and ~25% of the activity in another sample could not be accounted for by the inhibitors. Denitrosation of NDPA by three of the microsomal samples exhibited low Km values (51–86 µM) while two of these also had high Km values (2.6 and 4.6 mM). Purified human P450 2B6 and 3A4 and human P450 2A6, 2C8, 2C9 and 2D6 membranes had high Km values relative to their maximum turnover rates and are unlikely to participate in NDPA metabolism at micromolar concentrations. Conversely, purified rabbit 2E1 exhibited Km and Vmax values for depropylation of 52 µM and 13.4 nmol propionaldehyde/min/nmol P450, respectively. Values for denitrosation were 66 µM and 1.44 nmol nitrite/min/nmol P450, respectively. The toxicity of NDPA in transfected human liver epithelial cells expressing 2E1 was dose dependent down to 50 µM. No toxicity was observed in control cells or those expressing 2A6. These results indicate that 2E1 is the major human liver microsomal isoform responsible for NDPA metabolism at low micromolar concentrations. We also show that purified P450s catalyze the denitrosation of NDPA at ~10–20% of the rate of depropylation and Km values for both reactions are the same for each isozyme. This is consistent with the formation of an initial intermediate common to both pathways, presumably an {alpha}-nitrosamino radical.

Abbreviations: DDC, diethyldithiocarbamic acid; DLPC, dilauroylphosphatidylcholine; DNPH, 2,4-dinitrophenylhydrazine; ICl, intrinsic clearance; mAb, inhibitory monoclonal antibody; NDMA, N-nitrosodimethylamine; NDPA, N-nitrosodi-n-propylamine; P450, cytochrome P450; TAO, troleandomycin; THLE cells, transformed human liver epithelial cells.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitrosamines are a large class of compounds that may be a factor in the induction of some human cancers due to their environmental presence and high carcinogenic potency in animal models. N-nitrosodi-n-propylamine (NDPA), a dialkylnitrosamine, has been detected at low levels in various cheeses, cured meats, colored alcoholic beverages and at higher levels in the air of factories that process molded rubber (1). This compound is a potent carcinogen in many species, including the rat, inducing tumors in the liver as well as many other organs, and is therefore a suspected human carcinogen (2,3). Dialkylnitrosamines must first be activated in vivo and the most critical step in activation is the cytochrome P450 (P450)-mediated {alpha}-hydroxylation (4,5; Figure 1Go). This is believed to occur through the initial formation of a {alpha}-nitrosamino radical which recombines with the hydroxyl radical at the P450 active site (5,6). For NDPA the {alpha}-hydroxy compound would spontaneously decompose to form propionaldehyde and propyldiazohydroxide. The propyldiazohydroxide readily decomposes to the propyldiazonium ion, a highly electrophilic species that propylates DNA inducing various types of damage (79). P450s can also catalyze the denitrosation of dialkylnitrosamines, which is generally believed to be a detoxification pathway (10). With nitrosodimethylamine (NDMA), studies strongly suggest that denitrosation is predominantly an oxidative pathway in which the putative {alpha}-nitrosamino radical fragments to nitric oxide and an imine (6,11; shown in Figure 1Go for NDPA). Nitric oxide and the imine are non-enzymatically oxidized and hydrolyzed, respectively, to nitrite, an amine and an aldehyde. There is also evidence for a P450-mediated one electron reduction of various aromatic nitrosamines, resulting in the formation of nitric oxide and a secondary amine (12,13). Due to the similarity in structure with NDMA it is speculated that denitrosation of NDPA is an oxidative process, although there is no direct evidence for this. Since microsomal denitrosation of dialkylnitrosamines appears to occur at a rate that is ~10–30% of dealkylation (6,14,15), aldehyde formation is commonly measured to estimate the rate of activation, e.g. {alpha}-hydroxylation.



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Fig. 1. Proposed mechanism for cytochrome P450-mediated metabolism of NDPA. The putative {alpha}-nitrosamino radical intermediate undergoes oxygen rebound at the enzymes active site to form the {alpha}-hydroxynitrosamine (a) or fragments to nitric oxide and the imine (b).

 
It was previously shown in our laboratory that P450 2E1 and 2B1 are the isozymes responsible for depropylation of NDPA by rat liver microsomes (16). Concordantly, these isozymes were shown to metabolize NDPA to toxic and DNA-damaging species at low micromolar concentrations in primary rat hepatocyte cultures (17,18). Since 2E1 is highly conserved across species and exhibits a high degree of substrate specificity, it was thought that it might contribute to NDPA metabolism in humans. Correlations between NDPA depropylation and the specific activities of 2E1 and 3A have been reported in human liver microsomal samples (19), although the substrate concentration used was 4 mM, well above the expected physiological concentrations.

To better understand the risk that this carcinogen may pose to humans, the present study was initiated to identify potential human liver P450s that may contribute to NDPA depropylation and denitrosation. Several common approaches were used to identify the P450s contributing to the metabolism of NDPA, i.e. kinetic, chemical inhibition and inhibitory antibody studies with human liver microsomes (20). Also, kinetic studies were performed with purified P450s in a reconstituted system and Escherichia coli membrane preparations containing human P450s. Additionally, toxicity studies were performed in cell lines derived from human liver epithelial cells which had been transfected so that they expressed individual human P450s.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
NDPA, NDMA, semicarbazide HCl, N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilamide, sulfaphenazole, troleandomycin, diethyldithiocarbamic acid, NADP+, NADPH, glucose 6-phosphate, glucose 6-phosphate dehydrogenase (Type IX), bovine serum albumin, dilauroyl-L-{alpha}-phosphatidylcholine, L-{alpha}-dilauroyl- and L-{alpha}-dioleyl-sn-glycero-3-phosphocholines, phosphatidyl serine, catalase and glutathione were obtained from Sigma Chemical Co. (St Louis, MO). Butyraldehyde (99% pure) and propionaldehyde (97% pure) were obtained from Aldrich Chemical Co. (Milwaukee, WI). 2,4-Dinitrophenylhydrazine and zinc sulfate were obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ). HPLC grade acetonitrile and hexanes were from Fisher (Pittsburgh, PA). Tissue culture media and materials were obtained from Biofluids Inc. (Rockville, MD), except for epidermal growth factor, human plasma fibronectin, fetal bovine serum, L-glutamine, gentamycin and Hank's balanced salt solution, which were obtained from Gibco BRL (Grand Island, NY).

Preparation of human liver microsomes
Sections of frozen human liver samples were a gift from Dr F.P.Guengerich (Vanderbilt University, Nashville, TN). Microsomes were prepared as previously described (21) with minor modifications indicated here. The liver tissue was placed in buffer (100 mM potassium phosphate, pH 7.4, 1 mM EDTA and 150 mM KCl) on ice, minced and homogenized with a Polytron homogenizer using 10 s bursts at ~7000 r.p.m. Butylated hydroxytoluene was omitted from the wash buffer and microsomal pellets were resuspended in a buffer containing 100 mM potassium phosphate, pH 7.4, 1 mM EDTA and 0.25 M sucrose and stored at –80°C. Protein content was determined using the bicinchonic acid method (22) and P450 content was determined spectrally (23).

Inhibition of microsomal NDPA depropylation by isozyme-specific chemical inhibitors
The measurement of NDPA depropylation was based on the HPLC method of Farrelly (24), in which the propionaldehyde product is converted to its spectrophotometrically detectable 2,4-dinitrophenylhydrazone derivative. NDPA was dissolved in acetonitrile; this solvent was <0.5% in all reactions. Reactions were performed in Tris buffer, pH 7.4, containing 150 mM KCl, 10 mM MgCl2 (buffer A), 0.5 mg microsomal protein, NDPA, 3 mM semicarbazide (adjusted to pH 7.4) to trap propionaldehyde as its semicarbazone derivative, and an NADPH generating system consisting of 15 mM glucose 6-phosphate, 0.6 mM NADP+ and 1 U/ml glucose 6-phosphate dehydrogenase, in a final volume of 1 ml. Incubations containing the P450 mechanism-based inhibitor diethyldithiocarbamic acid (DDC) or the quasi-irreversible inhibitor troleandomycin (TAO), both at final concentrations of 20 µM, were pre-incubated for 15 min at 37°C in the presence of the NADPH generating system. The reactions were initiated by adding semicarbazide and NDPA, to final concentrations of 3 and 4 mM, respectively, and incubated for 30 min at 37°C in sealed amber vials. Reactions containing the competitive inhibitor sulfaphenazole (10 µM) were pre-incubated for 3 min with 0.15 mM NDPA and were initiated by adding the NADPH generating system. The incubations were quenched by adding 0.1 ml of a 2:5 mixture of 0.1 M semicarbazide and 50% zinc sulfate containing butyraldehyde as an internal standard. The vials were mixed and placed on ice for 20 min. After addition of 0.1 ml of a saturated solution of barium hydroxide the vials were centrifuged to precipitate the pellet. The supernatant (0.8 ml) was mixed in a two phase medium containing 1.1 ml of a saturated 2,4-dinitrophenylhydrazine (DNPH) solution in 1 N HCl and 1.5 ml hexane. After 35 min, 1 ml of the hexane layer was removed and mixed with 0.35 ml of acetonitrile by vortexing. The acetonitrile layer was removed and an aliquot (0.05 ml) was analyzed using a Waters HPLC equipped with a Rainin microsorb C18 reverse phase column (4.6x250 mm) fitted with an Upchurch in-line filter. The DNPH derivatives were separated isocratically using 55/45% acetonitrile/water and detected at 340 nm using a Gilson HM Holochrome detector. Control reactions omitted the inhibitor. Background levels of propionaldehyde were determined by running control reactions without the NADPH generating system. Since TAO and sulfaphenazole were dissolved in methanol, an equal volume of methanol was added to their respective control reactions. Standards contained known quantities of propionaldehyde in buffer A containing 3 mM semicarbazide. Inclusion of microsomes, the NADPH generating system, methanol or inhibitors or incubation for 30 min at 37°C did not alter the recovery of propionaldehyde. Assays were run in duplicate.

Denitrosation assays using human liver microsomes
Denitrosation of NDPA results in the formation of nitrite, which was determined using a colorimetric assay (10). Incubations contained buffer A, the NADPH generating system, 0.5–1.5 mg microsomal protein and NDPA in a final volume of 1 ml. All incubations contained the same concentration of acetonitrile, which was never greater than 0.5%. After a 2 min pre-incubation at 37°C, the reactions were initiated by adding the NADPH generating system and quenched 30 min later by adding a 50% zinc sulfate solution (25 µl). A 50 µl aliquot of a saturated barium hydroxide solution was added and the reactions centrifuged. To 0.9 ml of the supernatant was added 0.25 ml of sulfanilamide (0.1 M in 3 N HCl), followed by addition of 0.125 ml of N-(1-naphthyl)ethylenediamine dihydrochloride (2 mM in 6 N HCl). Samples were mixed and incubated in the dark for 30 min to allow the azo dye to develop, then measured spectrophotometrically at 540 nm using a SLM Aminco 3000 spectrophotometer. Background nitrite was determined by adding the microsomal protein to the reactions after they had been quenched. Standards containing known quantities of sodium nitrite instead of NDPA were run in the same manner as the samples and reactions were run in duplicate.

Metabolism assays using purified P450s
Human P450 3A4 and 2B6, rabbit P450 2E1 and rat NADPH-cytochrome P450 reductase (reductase) were expressed in E.coli and purified (2527). Rat P450 2B1 and cytochrome b5 were purified from rat liver microsomes (25,27). Purified 2E1, 2B6 and 2B1 were reconstituted with reductase in dilauroyl-L-phosphatidylcholine (DLPC) for 1 h on ice (26,27). Cytochrome b5 was added to reconstitution mixtures containing 2E1 and 2B6. Incubations contained P450 (0.025–0.1 nmol), cytochrome b5 (in 2E1 and 2B6 reactions) and reductase in the ratio 1:1:2, 400 µg DLPC/nmol P450, 125 U/ml catalase, NDPA, 0.2% acetonitrile, 50 mM potassium phosphate buffer, pH 7.4, containing 40 µg/ml bovine serum albumin (buffer B) or, for 2E1 denitrosation, buffer A in a final volume of 1 ml. Reactions were pre-incubated for 3 min at 37°C, initiated by adding 1 mM NADPH and run for 10 (2E1 and 2B1) or 30 (2B6) min. For a typical reconstitution of 3A4 the following components were added in the order indicated so that the final contents were: 50 µg of a 1:1:1 mixture of L-{alpha}-dilauroyl- and L-{alpha}-dioleyl-sn-glycero-3-phosphocholines and phosphatidyl serine, 500 µg cholic acid, 0.5 nmol 3A4, 1 nmol reductase, 0.5 nmol cytochrome b5 and 30 mM MgCl2. After 30 min at room temperature, 1 µmol glutathione, 500 U catalase and 50 mM HEPES buffer, pH 7.5, containing 20% glycerol, 0.5 m EDTA and 30 mM MgCl2 (reaction buffer) were added so that the final volume was 1 ml. Incubations contained an aliquot (0.08 ml) of the reconstitution mixture, 2.5% acetonitrile and NDPA in reaction buffer so that the final volume was 1 ml. To allow for dissolution of the higher concentrations of NDPA, 3A4 reactions were pre-incubated for 10 min at 37°C. Reactions were initiated by adding 1 mM NADPH and incubated for 15 min. Reactions were quenched and analyzed for product formation as described above for the microsomal assays. Standards for nitrite contained all of the reaction components used in the purified P450 assays. Control reactions omitted NADPH. 2E1, 3A4, 2B6 and 2B1 did not metabolize propionaldehyde at the substrate concentrations used in the assays. Reactions were run in duplicate.

NDPA depropylation by solubilized membranes containing human P450s
Co-expression of P450s 2A6, 2C8, 2C9 and 2D6 with reductase and solubilization of the E.coli membranes were performed as previously described (25). Reaction mixtures contained solubilized membranes (0.02–0.1 nmol P450), the NADPH generating system, 50 µg catalase, NDPA, 0.4% acetonitrile and buffer B in 1 ml. After a 2 min pre-incubation at 37°C, the reactions were initiated by adding the NADPH generating system, quenched 30 min later and analyzed for propionaldehyde formation as described above. Since 2C9 membranes were found to metabolize propionaldehyde to some extent (25% in 30 min at 10 µM propionaldehyde), these reactions were also performed in the presence of 1 mM semicarbazide, which increased the product yield by 19%. Control reactions omitted the generating system.

Inhibition of microsomal NDPA depropylation by isozyme-specific inhibitory antibodies
Human P450 inhibitory monoclonal antibodies (mAbs) were kindly provided by Dr H.V.Gelboin (NIH, Bethesda, MD). These antibodies were shown to be highly specific and strongly inhibitory to the target P450s (28). Reaction conditions were similar to those previously reported (28) but scaled down to conserve mAbs. Initial studies with the antibodies indicated that some of the preparations contained a component that eluted close to acetaldehyde on the HPLC chromatogram. Since this might have interfered with the assays for P450-mediated metabolism, it was removed from the mAb preparations by an overnight dialysis in a borate-buffered solution (200 mM sodium borate, 160 mM sodium chloride, pH 8). Reaction mixtures (0.25 ml) containing 62 µg of the indicated mAb and 62 pmol microsomal protein were incubated in buffer B at 37°C for 5 min. The depropylation reaction was initiated by adding 0.25 ml of buffer B containing 0.5 mM NDPA, 6 mM semicarbazide and the NADPH generating system. After incubation for 30 min, the reactions were quenched and proteins precipitated as described for the chemical inhibition studies. The supernatant (0.6 ml) was mixed as described above in the two phase medium (1.1 ml of DNPH solution and 1.2 ml of hexane) and the dinitrophenylhydrazone back-extracted into 0.25 ml acetonitrile. The acetonitrile layer was analyzed as described above on a Gilson HPLC equipped with a Gilson 115 UV detector. Control reactions contained all components and 62 µg anti-lysozyme mAb (HyHel) instead of the P450 mAbs. Background propionaldehyde was determined in reactions by omitting the mAb and the NADPH generating system. Standards contained known quantities of propionaldehyde in buffer B with 3 mM semicarbazide. Reactions were performed in duplicate.

Determination of kinetic constants
For kinetic analysis the reaction rates were determined under conditions in which product formation was linear with time and protein concentration. Km and Vmax values were calculated by linear regression analysis of the data from Lineweaver–Burke plots using the Enzyme Kinetics program, v.1.04 from Trinity Software (Plymouth, NH).

THLE cell culture conditions
Immortalized human liver epithelial cells (THLE) transfected with human P450s, provided by Dr K.Mace (Nestle Research Centre, Switzerland), were cultured in fibronectin/collagen-coated flasks in serum-free liver cell medium (29) and maintained in a humidified incubator at 37°C with a 3.6% CO2 atmosphere.

Cytotoxicity assays
Toxicity was determined by the crystal violet viability assay (30). Cells were seeded at 75 000 cells/plate on individual fibronectin/collagen-coated 35x10 mm plates. On the following day the medium was replaced with medium containing the nitrosamine and 0.5% acetonitrile and incubated for 3 days. After 72 h the medium was removed and the attached cells rinsed with phosphate-buffered saline and fixed with 0.4% formaldehyde for 1 h. After removal of the formaldehyde the cellular nuclei were stained by a 30 min incubation with 0.1% crystal violet aqueous solution. The plates were washed with water and air dried. A 5% solution of SDS in water was added and after 30 min the absorbance of the solution was measured spectrophotometrically at 550 nm on a SLM Aminco 3000 spectrophotometer. Viability is proportional to the extent of staining by crystal violet. Assays with each concentration of NDPA were performed in triplicate. Data were analyzed by Student's t-test to determine statistical differences of the means.


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Depropylation of NDPA by human liver microsomes
Based on a previous study with various long chain nitrosamines, it was estimated that a substrate concentration of 4 mM would be near saturating in human liver microsomes (14). Therefore, this concentration of NDPA was used to approximate maximum depropylation rates in seven human liver microsomal samples. The rates ranged from 0.53 to 2.60 nmol propionaldehyde/min/mg protein (average 1.15 nmol/min/mg). These rates are comparable with those reported for microsomes from uninduced rats, 2.0 and 2.7 nmol/min/mg (16,31). In a previous study with human liver microsomal samples a somewhat lower average depropylation rate of 0.35 nmol/min/mg and range of 0–0.92 (n = 14) was reported (19). Lower rates in this earlier study, in which semicarbazide was not added to trap propionaldehyde, are most likely due to microsomal degradation of the product, which we found to occur at an appreciable rate in the absence of semicarbazide (data not shown).

Inhibition of NDPA depropylation in microsomal samples by specific chemical inhibitors of various P450s
Microsomal NDPA depropylation rates were determined in the presence of isozyme-specific chemical inhibitors. The choice of inhibitors for these studies was based on Bellec's correlation studies in human microsomes which suggested that 2E1 and 3A4/5 were involved in the depropylation reaction at saturating substrate concentrations (4 mM) (19). In these assays we used this concentration of NDPA to see if our results would agree with those of Bellec and to initially identify contributing P450s that could be studied further. Figure 2Go shows the extent of inhibition of depropylation by 20 µM DDC and TAO. Since both of these are irreversible or quasi-irreversible inhibitors and they were pre-incubated with the microsomes prior to substrate addition, the extent of inhibition should not be significantly diminished by substrate competition. DDC displayed a marked inhibition in all the samples and almost completely inhibited the reaction in samples 132 and 129. This concentration of DDC is very effective at inhibiting 2E1 activity, although it has also been shown to be a less potent inhibitor of other P450s such as 2A6 and 2C19 (32,33). TAO is highly specific for the 3A isozymes at a concentration of 20 µM (32) and inhibited depropylation in sample 110 by >50%, while inhibition in the rest of the samples ranged from ~0 to 20%. No significant inhibition was apparent when 10 µM sulfaphenazole, a potent 2C9 competitive inhibitor, was used with 0.15 mM NDPA (data not shown). Three of the liver samples in our study had previously been characterized for specific P450 activities and the results are shown in Table IGo (34,35). Our inhibition results are consistent with their findings, e.g. the extent of inhibition by an isozyme-specific inhibitor corresponded to the level of activity of the isozyme in the sample.



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Fig. 2. Inhibition of human liver microsomal NDPA depropylation by the P450 isozyme-specific chemical inhibitors DDC (2E1, 2A6 and 2C19) and TAO (3A). Values are the average of two separate experiments done in duplicate. Error bars represent the range of the means.

 

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Table I. P450 isozyme-specific activities determined previously by Tateishi et al. (34) in human liver microsomal samples
 
Inhibition of human liver microsomal depropylation of NDPA by inhibitory mAbs
The ability of isozyme-specific inhibitory mAbs to inhibit NDPA depropylation in four of the microsomal samples was also determined. In these studies we used NDPA at 0.25 mM so that the contribution by isozymes with low affinities would be minimized. Concentrations lower than 0.25 mM were not used because of concerns regarding product yields that would be below the detection limits. As seen in Figure 3Go, inhibition by the 2E1 mAb was >50% in all four samples and very similar to the inhibition profile seen with DDC, suggesting that 2E1 is the predominant isozyme responsible for depropylation at this sub-saturating substrate concentration. Little to no inhibition by the 2A6, 3A4 and 2D6 mAbs suggests that these isozymes are not significantly involved in NDPA depropylation at the substrate concentration tested. The 2C mAb, which is specific for 2C8/9/18/19, did show significant inhibition, ~25%, in sample 136. Since the depropylation activity of 2C8 and 2C9 membrane preparations was very low at 0.2 mM NDPA (Table IIGo) and no inhibition was seen in the microsomes with sulfaphenazole, a potent inhibitor of 2C9, the isozyme(s) inhibited by the mAb in this sample is probably 2C18 and/or 2C19. Approximately 25% of the activity in sample 39 could not be accounted for using the mAbs or the chemical inhibitors.



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Fig. 3. Inhibition of human liver microsomal NDPA depropylation by inhibitory mAbs specific for P450s 2E1, 2C, 2A6, 2D6 and 3A4. Values are the average of two separate experiments done in duplicate (except for sample 114, which was only done in duplicate with the 2E1 mAb). Error bars represent the range of the means.

 

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Table II. Depropylation of NDPA by solubilized E.coli membranes containing various P450sa
 
Depropylation of NDPA by solubilized E.coli membranes containing various human P450s
The potential of isozymes other than those identified by the chemical inhibition studies to metabolize NDPA was investigated using solubilized E.coli membranes co-expressing various P450s and P450 reductase. P450s and P450 reductase are membrane proteins and when expressed in E.coli they are inserted into the bacterial outer membrane and retain their catalytic activities. Many studies have shown that catalytic activities determined using these preparations can be comparable with values determined using purified–reconstituted systems or human liver microsomes (3639). Therefore, the membranes were used to provide an estimate of the ability of various P450s to depropylate NDPA. All four P450s tested could catalyze the depropylation reaction at the estimated saturating NDPA concentration of 4 mM (Table IIGo). When the concentration was lowered to 0.2 mM, depropylation by 2A6 was 0.8 nmol propionaldehyde/min/nmol P450, still greater than half the maximum rate of 0.65. This indicates that the Km for 2A6 is below 0.2 mM. Of the other three isozymes only 2D6 could significantly catalyze the reaction at 0.2 mM NDPA. The rate of the reaction, 0.24 nmol propionaldehyde/min/nmol P450, suggests that this concentration is below the Km value for 2D6.

NDPA metabolism by purified P450s
Km and Vmax were determined for NDPA metabolism using purified P450 isozymes in the reconstituted system and the intrinsic clearance (ICl), the ratio Vmax/Km, was calculated. An isozyme(s) with a higher ICl will be most effective at catalyzing the reaction at low sub-saturating substrate concentrations, although microsomal levels of the isozymes must also be considered when establishing the contribution of a particular P450. The P450s could catalyze both denitrosation and depropylation of NDPA, with the former being ~10–20% of the latter (Table IIIGo). Also, Km values for each P450 were the same, within experimental error, for both reactions. Maximum metabolic rates for denitrosation and depropylation by 3A4 were high, although the Km values were high as well, resulting in a relatively low ICl. Rat 2B1 has previously been shown to contribute significantly to the metabolism and activation of NDPA in this species (16,17). For comparative purposes, kinetic constants for NDPA metabolism by 2B1 were determined. Km values for denitrosation and depropylation were 100 and 103 µM, respectively, with relatively high Vmax values of 5.7 and 27.5 nmol/min/nmol, respectively. Kinetic constants for metabolism by purified rabbit 2E1 were also determined. Like rat 2E1, the rabbit isoform has a very high sequence homology and overlapping substrate specificity with human 2E1 (40,41). Since rabbit 2E1 was readily available in our laboratory, we used it in lieu of the human isozyme. The Km values for depropylation and denitrosation were 52 and 66 µM, respectively. 2E1 also exhibited appreciable Vmax values of 1.4 and 13.4 nmol/min/nmol for denitrosation and depropylation, respectively. The ICl values of 2E1 and 2B1 were reasonably comparable and at least 25 times greater than that of 3A4. P450 2B6, the closest human ortholog to 2B1, was much less efficient at catalyzing depropylation than 2B1 or 2E1 and exhibited an ICl value for depropylation of 3.0, almost 100 times lower than that of 2B1. Denitrosation assays were not performed with 2B6 due to its low activity and the expectation that the product yields would be below the detection limit.


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Table III. Kinetic constants for the metabolism of NDPA by purified P450sa
 
Denitrosation of NDPA by human liver microsomes
In order to characterize in more detail the ability of the human microsomes to metabolize NDPA, we determined the kinetic constants for NDPA denitrosation by three of the samples. Although the constants for depropylation are generally considered to be more relevant to activation, these constants could not be accurately determined in human microsomes due to degradation of the propionaldehyde product by the microsomes, as mentioned above. Additionally, semicarbazide could not be added to trap the aldehyde product and prevent its further metabolism since it is a potent competitive inhibitor of 2E1 (42), which has been identified as one of the major contributors to NDPA metabolism here and in previous studies (16). Since denitrosation by P450s appears to be a constant proportion of depropylation (Table IIIGo), microsomal rates of this reaction can be used as an estimate of activation. All three samples could readily catalyze NDPA denitrosation (Table IVGo). Samples 110 and P4, which showed significant inhibition of depropylation by both DDC and TAO, exhibited biphasic denitrosation kinetics (shown in Figure 4Go for sample 110). This indicates that there are at least two isozymes responsible for NDPA denitrosation in these samples. The Km values for the high affinity component catalyzing the denitrosation were 86 and 51 µM, similar to the values for denitrosation and depropylation by purified 2E1, and values for the low affinity component were 2.6 and 4.6 mM for samples 110 and P4, respectively (Table IVGo). Figure 4Go also shows the monophasic denitrosation kinetics exhibited by sample 132, in which depropylation was almost completely inhibited by DDC and the 2E1-specific mAb. The Km value for this sample was 59 µM, similar to the low Km values in the other two samples and those of purified 2E1.


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Table IV. Kinetic constants for denitrosation of NDPA by human liver microsomal samplesa
 


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Fig. 4. Eadie–Hofstee plots of NDPA denitrosation kinetic data for human liver microsomal samples 110 and 132. Values are the average of two separate experiments done in duplicate. Lines represent the least squares fit of the data.

 
Cytotoxicity of NDPA in THLE cells expressing P450s
The ability of human P450s to catalyze the metabolic activation of NDPA to highly reactive toxic forms was examined in human cells expressing P450s. These THLE cell lines exhibit high levels of the specific transfected human P450 and have been shown to retain many of the phase II enzymes (29,43). Viability of the cells was determined using the crystal violet staining method, which is a general measure of cytotoxicity. Figure 5Go shows the viability of THLE cells expressing human 2E1 exposed to 0–2 mM NDPA or NDMA, a potent liver toxicant and carcinogen activated by 2E1 to an alkylating species (44). Both compounds showed a dose-dependent cytotoxicity for the 2E1-expressing cells, with toxicity leveling off at ~60 and 20% relative survival for NDPA and NDMA, respectively. The lowest concentration tested, 50 µM, still showed a statistically significant decrease in viability with both compounds. No toxicity was observed in control THLE cells not transfected with P450s or THLE 2A6 cells with nitrosamine concentrations up to 4 mM (data not shown).



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Fig. 5. Dose–response curves for NDPA and NDMA cytotoxicity in THLE cells expressing human P450 2E1. NDPA ({square}) or NDMA (•) was added to the culture medium at the concentrations indicated and relative survival was determined as described in Materials and methods. Data are the means ± SD from representative experiments with each point determined in triplicate. All treated samples were significantly different from the untreated controls (P < 0.05 by Student's t-test).

 

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 Abstract
 Introduction
 Materials and methods
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 Discussion
 References
 
NDPA is a potent rat carcinogen and P450s 2E1 and 2B1 have been shown to be the major isozymes responsible for its activation in the liver of this species (1618). Our study uses several complementary in vitro approaches to implicate P450 2E1 as the predominant human liver microsomal isozyme catalyzing depropylation, which reflects the rate of {alpha}-hydroxylation, and denitrosation of NDPA at low micromolar concentrations. Purified rabbit 2E1, which has a similar substrate specificity to human and rat 2E1, had Km values of 53 and 66 µM for depropylation and denitrosation, respectively, and significant Vmax values, resulting in relatively high ICl values. At 4 mM NDPA our human microsomal samples effectively catalyzed depropylation and exhibited significant inhibition by the potent 2E1 chemical inhibitor DDC. Four samples that were also examined with inhibitory mAbs showed that depropylation was largely inhibited, ~60–90%, by the 2E1 mAb at 0.25 mM NDPA. Km values between 51 and 86 µM reported for denitrosation in the three microsomal samples tested are similar to those for denitrosation and depropylation by purified 2E1. This concurs with the observation of a significant inhibition of depropylation by DDC in these three samples, with one sample also exhibiting almost complete inhibition by the 2E1 mAb. The ability of human 2E1 to metabolize NDPA to a toxic species, presumably through the {alpha}-hydroxylation pathway, at micromolar concentrations was confirmed in THLE 2E1 cell cultures.

P450s 2C18 and/or 2C19 may also partly contribute to the metabolism of NDPA at 0.25 mM in some individuals, as the 2C mAb inhibited depropylation in sample 136 (~25%). About 25% of the depropylation in sample 39 could not be accounted for based on inhibition by the mAbs or the chemical inhibitors. This indicates that unidentified P450s may also contribute to depropylation at the substrate concentrations tested, although it is possible that inhibition by the mAbs or chemical inhibitors is incomplete. P450s 1A1 and 1A2 were not investigated because no correlation between NDPA depropylation and these specific activities was seen in the study by Bellec et al. (19). Also, previous results suggested that rat 1A1 and 1A2 do not participate in NDPA depropylation (16).

Bellec et al. (19) found 2E1 and 3A4 to be the major isoforms involved in NDPA depropylation in human liver microsomes at substrate concentrations estimated to be saturating. Our results agree with and extend theirs, identifying the P450s mentioned above as contributors to NDPA metabolism at micromolar concentrations. To understand the risk NDPA poses as a human carcinogen, determination of the metabolism of NDPA at physiological concentrations is critical. These concentrations are difficult to establish due to the vast array of possible sources of this compound. NDPA can potentially form when secondary or tertiary amine-based compounds come into contact with nitrosating agents (e.g. oxides of nitrogen) and this has been implicated as the source of low parts per billion levels of NDPA in some foodstuffs and industrial settings (4548). A recent survey of the gastric juice of 71 patients found NDPA levels of 0.10 ± 0.57 nmol/l (49), indicating that exposure of the general population may be low, yet potentially widespread. Other individuals may be at risk for higher exposures. One study detected NDPA in the atmosphere of a rubber production factory at levels of 1.3– 3.3 µg/m3 (48). Due to the limitations of our analytical methods, we could not measure metabolism at sub-micromolar concentrations. However, kinetic constants calculated for 3A4 and 2B6 and those estimated for 2C8, 2C9, 2A6 and 2D6 indicate that their Km values are high relative to their turnover numbers, indicating that they are unlikely to catalyze depropylation at physiological concentrations. It can be inferred from its ICl that 2E1-mediated depropylation will occur at high nanomolar concentrations, although activity may be insignificant at lower concentrations. 2E1 is constitutively expressed and highly inducible by alcohol (50), suggesting that activation of NDPA may be a concern if exposure is high enough. Further investigation of metabolism at lower concentrations with tritium-labeled compounds, as has been done with methyl-n-amylnitrosamine and tobacco-specific nitrosamines (51,52), will provide a better understanding of the carcinogenic risk of NDPA.

Generally, the rates of microsomal denitrosation of dialkylnitrosamines occur at ~10–20% of the rates of dealkylation, suggesting a close association between the two pathways (6,14,15, and results herein). Of the three purified P450s examined here, all catalyzed denitrosation of NDPA at a rate of 10–20% that of dealkylation (Table IIIGo). Purified 2B1 was also previously shown to denitrosate nitrosomethylamylamine at ~20% of the rate of dealkylation (15). These findings conclusively demonstrate that individual P450s catalyze both reactions and suggest that this may be common for many dialkylnitrosamines. Mechanistically, studies with deuterium-labeled NDMA have suggested that P450 oxidizes the nitrosamine to an initial {alpha}-nitrosamino radical intermediate which undergoes oxygen rebound to the {alpha}-hydroxy compound or fragments to nitric oxide and the imine (6). Non-enzymatic generation of the {alpha}-nitrosamino radical from NDMA by the Fenton reagent also supports this pathway (11). The same Km values for denitrosation and depropylation of NDPA by each P450 examined (Table IIIGo) are consistent with this oxidative mechanism. A P450-mediated reductive denitrosation mechanism, as mentioned above, for NDPA cannot be ruled out, although disparate pathways of oxidative depropylation and one electron reductive denitrosation are much less likely to exhibit equivalent Km values.

NDPA can also be metabolized to a species capable of methylating as well as propylating nucleic acids in rat livers in vivo (53). In addition, P450s can oxidize this compound at the ß and {omega} positions, possibly leading to chain shortened derivatives (16,54). The extent of these and other biotransformations in human tissues and the effect they may have on tumor initiation is not known. The enzymes involved in the metabolism of NDPA to a methylating species are under investigation.


    Notes
 
3 To whom correspondence should be addressed Email: phollen{at}umich.edu Back


    Acknowledgments
 
We thank Dr F.P.Guengerich for the human liver samples, Dr H.V.Gelboin for the mAbs and Drs K.Mace and A.M.A.Pfeifer for the THLE cells. We also thank Dr J.R.Reed for many helpful discussions. This work was supported in part by NIH grant CA-16954 to P.F.H.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 8, 1999; revised April 28, 2000; accepted May 3, 2000.