Metabolism of the ß-oxidized intermediates of N-nitrosodi-n-propylamine: N-nitroso-ß-hydroxypropylpropylamine and N-nitroso-ß-oxopropylpropylamine

John F. Teiber1,2, Katherine Macé3 and Paul F. Hollenberg2,4

1 Department of Environmental and Industrial Health and
2 Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-0632, USA and
3 Nestlé Research Center, PO Box 44, CH-1000, Lausanne 26, Switzerland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The rat liver carcinogen N-nitrosodi-n-propylamine (NDPA) is metabolized to a propylating and methylating species in vivo. Metabolism to a methylating species is believed to require an initial hydroxylation by cytochrome P450s (P450s) to N-nitroso-ß-hydroxypropylpropylamine (NHPPA), which is oxidized to N-nitroso-ß-oxopropylpropylamine (NOPPA), followed by a P450-mediated depropylation to ß-oxopropyldiazotate, which non-enzymatically breaks down to the methylating agent. Purified rat liver P450 2B1 and rabbit liver 2E1 in the reconstituted system and liver microsomes from phenobarbital (PB) and pyridine (Pyr) treated rats readily metabolized NOPPA to a methylating species as determined by the in vitro formation of 7-methylguanine (m7Gua) in DNA. Exposure of cells derived from the human liver epithelium transfected with human 2E1 (T5-2E1) to NOPPA resulted in the formation of m7Gua DNA adducts and a dose dependent toxicity. In vitro incubation of NHPPA with microsomes from PB, Pyr and non-treated (NT) rats and a human microsomal sample also resulted in m7Gua formation. P450s 2B1 and 2E1 oxidized NHPPA to NOPPA, forming 16.5 ± 3.1 and 20.0 ± 4.4 pmol NOPPA/pmol P450 in 1 h, respectively. Rat liver cytosol, in the presence of NAD+, oxidized NHPPA to NOPPA at a rate of 13.7 ± 3.0 pmol/min/mg protein while microsomes from NT rats catalyzed this reaction at 95.6 ± 16.5 pmol/min/mg protein. Cells derived from hamster lung tissue (V79 control) and T5-neo cells oxidized NHPPA to NOPPA. This oxidation was about 15 fold higher in T5-2E1 or V79 cells transfected with human 2E1 or rat 2B1, respectively. The results are consistent with the putative sequential oxidation pathway and suggest that, at the concentrations tested, oxidation of NHPPA to NOPPA may be predominantly mediated by cytochrome P450s. In addition, it appears that rabbit, rat and human P450 2E1 can catalyze both oxidations.

Abbreviations: DLPC, dilauroyl-L-{alpha}-phosphatidylcholine; GS, NADPHgenerating system; m7Gua, 7-methylguanine; NDMA, N-nitrosodimethylamine; NDPA, N-nitrosodi-n-propylamine; NHPPA, N-nitroso-ß-hydroxypropylpropylamine; NMPA, N-nitrosomethylpropylamine; NOPPA, N-nitroso-ß-oxopropylpropylamine; NT, non-treated; P450, cytochrome P450; PB, phenobarbital; Pyr, pyridine; reductase, NADPH-cytochrome P450 reductase; T5 cells, transfected T-antigen immortalized human liver epithelial cells; TLC, thin layer chromatography; V79 cells, hamster lung tissue cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-Nitrosodi-n-propylamine (NDPA), a dialkylnitrosamine, is a potent animal carcinogen, inducing primarily liver, esophageal and nasal cavity tumors in the rat (1,2). This compound is ubiquitous in the environment, having been detected at low levels in various foodstuffs, herbicides and in rubber production factories (reviewed in 3). The critical step in activation of dialkylnitrosamines is believed to be the cytochrome P450 (P450)-mediated hydroxylation at the C{alpha} position (4). The {alpha}-hydroxy compound is unstable, releasing an aldehyde and forming a diazohydroxide (or diazotate in the deprotonated state) which spontaneously decomposes to form the electrophilic alkyldiazonium ion (5,6). The alkyldiazonium ion alkylates macromolecules, including DNA, leading to various types of damage such as premutagenic lesions and chromosomal aberrations, which are critical in the tumor initiation process (reviewed in 7). As expected from the scheme described above, P450-mediated {alpha}-hydroxlation of NDPA results in the formation of propionaldehyde and the propylating species (5,8). After administration of NDPA to rats, propylated DNA adducts were detected; however, the major adduct was 7-methylguanine (m7Gua) (9). Methylating agents are well known to be highly toxic and mutagenic (reviewed in 10) and methylation is likely to be contributing to the carcinogenicity of this compound. Since NDPA and many of its oxidized derivatives are commonly used to induce and study the tumorigenic process (11,12), a detailed understanding of the metabolism of NDPA and it derivatives is necessary to fully understand the mechanism by which they induce tumors. Additionally, this information will help to determine the carcinogenic risk that NDPA poses to humans. Due to the similarity in the structures of dialkylnitrosamines, investigation of the metabolism of NDPA will also aid in elucidating potential metabolic routes that other long chain nitrosamines may take.

Current evidence supports a consecutive ß-oxidation pathway, first proposed by Kruger (9) and subsequently modified by Leung and Archer (13; Figure 1Go), as the predominant pathway by which NDPA is converted to a methylating species in the rat liver. Initial support for this pathway came from the observation that administration of N-nitroso-ß-hydroxypropylpropylamine (NHPPA) or N-nitroso-ß-oxopropylpropylamine (NOPPA) to rats resulted in increased formation of m7Gua adducts in the liver, with the methyl groups being derived from the {alpha}-carbons of the hydroxypropyl and oxopropyl chains, respectively (14,15). These putative ß-oxidized intermediates were also shown to be potent liver carcinogens in the rat and mouse and were subsequently detected in rat urine after administration of NDPA (1618). P450-mediated hydroxylation of long chain nitrosamines at carbons other than the {alpha}-position is common and rat and human liver microsomes as well as purified–reconstituted human P450 2E1 have been shown to catalyze the ß-hydroxylation of NDPA (1921). The second reaction in the pathway, oxidation of NHPPA to NOPPA, was shown to occur in the presence of rat liver microsomes (22), although the enzyme(s) involved and the ability of other cellular fractions to catalyze this reaction have not been investigated previously. Kruger initially speculated that NOPPA could undergo deacetylation to form N-nitrosomethylpropylamine (NMPA), a potent rat liver carcinogen which is activated to a methylating species by P450-mediated {alpha}-hydroxylation on the propionaldehyde chain (16,23). However, formation of NMPA from NOPPA could not be detected in vivo or in vitro with various rat liver fractions nor could it be detected in vivo or in primary rat liver hepatocytes after exposure to NDPA (18,24,25). As an alternative route for the formation of a methylating species, Leung and Archer (13) hypothesized that, as with dialkylnitrosamines in general, NOPPA could undergo P450-mediated {alpha}-hydroxylation on the propyl chain, forming the oxopropyldiazotate following the spontaneous release of propionaldehyde (Figure 1Go). Their investigations have shown that the oxopropyldiazotate can methylate DNA in vitro and does form diazomethane. This is thought to occur by an internal nucleophilic attack by the diazotate oxygen on the carbonyl carbon to yield a cyclic oxadiazoline, which would then break down to acetic acid and the methylating species diazomethane (13). It was subsequently shown that NOPPA is metabolized to a DNA methylating species in vitro by a microsomal, P450-dependent oxidase(s) from the rat liver (26). Significantly, no formaldehyde was detected during the microsomal depropylation of NOPPA. This suggests that microsomal deacetylation to NMPA, which could then be deformylated, does not readily occur in the microsomal incubations, further ruling out this compound as the proximate methylating nitrosamine intermediate. However, NMPA formation from NOPPA or NHPPA by other cellular fractions or in vivo cannot be ruled out since NMPA is efficiently metabolized and may be a very transient species (23).



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Fig. 1. Sequential oxidation of NDPA to a methylating species.

 
Using cellular fractions from the rat and human liver, purified P450s and two different cell lines, we have identified enzymes involved in the oxidation of NHPPA to NOPPA and in the metabolism of NOPPA to a methylating species.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
NDPA, NDMA, NMPA, 7-methylguanine, calf thymus DNA, NADP+, NAD+, NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase (type IX), dilauroyl-L-{alpha}-phosphatidylcholine, L-{alpha}-dilauroyl- and L-{alpha}-dioloyl-sn-glycero-3- phosphocholines, phosphatidyl serine, catalase and glutathione were obtained from Sigma Chemical (St Louis, MO). Fluorescamine was obtained from Pierce (Rockford, IL). Solvents were spectroscopic grade or better obtained from Fisher (Pittsburgh, PA). Tissue culture media and materials were obtained from Biofluids (Rockville, MD) except for epidermal growth factor, human plasma fibronectin, fetal bovine serum, L-glutamine, gentamycin, Hanks balanced salt solution, Dulbecco's modified Eagle's media (DMEM), penicillin, streptomycin and G418 which were obtained from Gibco BRL (Grand Island, NY). All other chemicals were reagent grade or better obtained from commercial sources.

Enzymes
Rabbit cytochrome P450 2E1 and rat NADPH-cytochrome P450 reductase (reductase) were expressed in Escherichia coli and purified as described (27). Rat cytochrome P450 2B1 and cytochrome b5 were purified from rat liver microsomes as described (28,29).

Preparation of liver microsomes and cytosol
Frozen human liver samples were a gift from Dr Paul Watkins (University of Michigan, Ann Arbor, MI). Human microsomes were prepared as previously described (30) and stored in suspension buffer (100 mM potassium phosphate pH 7.4, 1 mM EDTA and 0.25 M sucrose) at –80°C. Microsomes were prepared from male Long Evans rats and cytosol was prepared from male Fischer-344 rats as described previously (8,31). Microsomes from non-treated (NT) and pyridine (Pyr) treated rats were dialyzed over night in suspension buffer before use to remove glycerol. Protein contents were determined using the bicinchonic acid method (32) and cytochrome P450 contents were determined spectrally (33).

Synthesis of NHPPA and NOPPA
NHPPA was synthesized and distilled as previously described (15) with minor modifications described here. The 2-hydroxy-propyl-propylamine HCl solution was not evaporated in vacuo but evaporated overnight in a glass tray to leave the crude residue. The NHPPA distillate was further purified by thin layer chromatography (TLC) on PK6F silica gel 60 A plates, 20x20 cm2 at 500 µm layer thickness (Whatman) using as a developing solvent 100:70:50 dicholoroethane:ether:hexanes. The band containing NHPPA was scraped off the plate and extracted into ether by vortexing. The extract was filtered using a 0.5 µm PTFE syringe filter unit (Millipore, Milford, MA) and the ether evaporated to leave the product. The identity of NHPPA was confirmed by elemental analysis and mass spectrometry using chemical ionization. The major ion had a mass of 147, which corresponds to the mass of NHPPA plus a hydrogen ion. Purity was confirmed by HPLC analysis on a Gilson instrument equipped with a Rainin C18 Microsorb-MV 100 A column and UV detector (254 and 340 nm). The mobile phase was a 10% acetonitrile solution run at 0.8 ml/min. The NHPPA was separated into its two geometric isomers, which appeared to be quantitatively equal, and had retention times of approximately 22.0 and 23.2 min. No other peaks were detectable on the HPLC chromatograms.

NOPPA was synthesized by oxidizing the NHPPA (34). A solution of 706 mg KCr2O7, 0.525 ml 36MH2SO4 and 3.5 ml water was added over a 30 min time period to 1 ml of crude NHPPA in 2.8 ml ether stirring at 25°C. After 1.5 h the ether layer was removed and its volume reduced by drying under nitrogen. The crude NOPPA was partially purified by TLC as described above and then re-purified by TLC, except the ratio of the solvents in the developing mixture was 5:30:25. The identity and purity of the NOPPA was confirmed by GC/MS with chemical ionization. The NOPPA eluted as one large peak (estimated to be roughly 99% of the total area) with a major ion of 145 mass units, corresponding to the mass of NOPPA plus a hydrogen ion.

The purity of NOPPA was also determined by HPLC under the conditions described above for NHPPA. The NOPPA was separated into its two geometric isomers, exhibiting retention times of approximately 23.7 and 27.9 min. No other peaks were detectable on the HPLC chromatogram. Based on the area of the two peaks, the amount of the isomer with a retention time of 23.7 min was present at approximately 10-fold the amount of the other isomer. It has been reported that the E isomer of N-nitroso-N-methyl-(2-oxopropyl)amine is metabolized by rat hepatocytes while the Z isomer is not (35). Since the preferential metabolism of one of the isomers of NHPPA or NOPPA by the enzymes identified in this study is possible, the cited concentrations of these nitrosamines may not reflect the concentration of the isomeric form metabolized. NHPPA and NOPPA were stored at –80°C. Their purity was periodically checked by TLC.

DNA isolation and analysis
DNA was isolated from in vitro reactions or cells and purified using the Cell Culture Midi kit (Qiagen) following the manufacturer's instructions except that the DNA remaining in the first flow through the column was re-isolated on the regenerated column and combined with the DNA solution which was isolated initially. The DNA pellet was dissolved overnight at room temperature in 100 µl water and the concentration and purity determined from the UV absorbances at 260 and 280 nm. M7Gua was liberated by heating the DNA at 100°C for 35min in 5mM KPi buffer (pH 7.4) (36). The samples were centrifuged for 10 min using an Eppendorf 5412 micro-centrifuge (Brinkman, Westbury, NY) and the supernatant removed and filtered through a PVDF 0.45 µm syringe filter unit (Millipore, Milford, MA). A diluted aliquot of the filtrate was injected onto a Gilson HPLC system containing tandem reversed-phase C18 columns (Rainin Microsorb). The mobile phase, 50 mM KPi (pH 5.5) containing 10% methanol, was filtered through a 0.22 µm filter (Millipore, Milford, MA) and sonicated. The flow rate was 0.8 ml/min. M7Gua was detected on an ESA (Bedford, MA) electrochemical detector (set at +0.8 V) equipped with a 5011 analytical cell. M7Gua co-eluted with known standards that had been boiled, centrifuged and filtered in the same manner as the samples.

In vitro formation of m7Gua by purified P450s and microsomes
Purified P450 2E1 or 2B1 were reconstituted with reductase, cytochrome b5 (only in 2E1 reconstitutions) in ratios of 1:2:1 and dilauroyl-L-{alpha}-phosphatidylcholine (DLPC) for 45 min on ice followed by the addition of catalase (27,29). The reconstituted mixtures were diluted to an appropriate volume with the phosphate buffer and added to the incubations so that the final concentrations of DLPC and catalase were 200 µg/ml and 100 U/ml, respectively. Incubations contained 50 mM KPi (pH 7.4), 40 µg/ml BSA, the NADPH-generating system (GS) (0.8 mM NADP+, 20 mM glucose-6-phosphate, 1 U glucose-6-phosphate dehydrogenase) 0.5 mg calf thymus DNA, 1 mM nitrosamine and microsomal protein (1–7 mg) or the purified–reconstituted P450 mixture (0.75–2 nmol P450) in a total volume of 1 ml. Stock NOPPA solutions were added in acetonitrile, which was never more than 0.2% final concentration in the incubation mixture. Incubations were placed in a shaking water bath (37°C) and initiated by adding GS. Control incubations omitted either the nitrosamine or GS. DNA was isolated and analyzed as described above.

Cell culture conditions
Immortalized human liver epithelial cells (T5) were cultured on fibronectin/collagen coated flasks in serum free liver cell medium (Biofluids, Rockville, MD) (37). V79 cells were provided by Dr J.Doehmer (Technische Universitat Munchen, Munchen, Germany) and cultured in DMEM (high glucose) supplemented with 5% FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin. Media for the V79 2B1 cells also contained 200 µg/ml G418. Cells were maintained in a humidified incubator at 37°C with a CO2 atmosphere of 3.6% for T5 cells and 5% for V79 cells.

M7Gua formation in T5 cells
T5 cells were grown to near confluence in 75 cm2 flasks and the media replaced with 10 ml media containing the appropriate concentration of nitrosamine. After 12 h the cells were detached and the DNA isolated using the Qiagen Cell Culture Midi kit and analyzed as described above.

Cytotoxicity assays using T5 cells
Toxicity was determined using the crystal violet viability assay (38) as previously described (30). Assays with each concentration of nitrosamine were performed in triplicate and data were analyzed by the Student's t-test to determine statistical differences in the means.

Oxidation of NHPPA to NOPPA in T5 and V79 cells
Medium (2 ml) containing the appropriate concentration of NHPPA was added to 75 cm2 flasks containing cells grown to near confluence or flasks without cells. After 23 h, the medium was removed, the flasks rinsed with 2 ml Hank's balanced salt solution and the cells counted (each flask contained approximately 20x106–40x106 cells). For each flask, the rinse was combined with the medium. Ethyl acetate (2 ml) was added to the combined medium and rinse. The mixture was thoroughly vortexed, centrifuged and the ethyl acetate layer removed. The extraction was repeated two more times, except only 1 ml ethyl acetate was added each time, and the combined ethyl acetate aliquots were then dried under a stream of nitrogen. The remaining precipitate was dissolved in methanol and spotted on a TLC plate. Standards were spotted directly onto the plate. The plates were developed using the mobile phase of 100:70:50 dicholoroethane:ether:hexanes. After drying, the plates were exposed to short wave UV irradiation and stained with fluorescamine as previously described (39). Under long wave UV irradiation, a digital image of the plate was generated with a Kodak digital camera, model EDAS 120, and the spots were quantitated by densitometry of the image using the Image 1.49 software program (National Institutes of Health). The standard curve for NOPPA was log linear as described (39) and the detection limit was approximately 80 pmol. No NOPPA was detected in flasks that contained 0.5 mM NHPPA without cells. To determine percent recovery of NOPPA, medium from flasks containing cells was spiked with 0.5 mM NHPPA and varying concentrations of NOPPA and then analyzed as described above. Quantitation of NOPPA was based upon the percent recovery, which was always between 70 and 90%.

Oxidation of NHPPA to NOPPA in vitro
Incubations with purified P450s, reconstituted as described above, or microsomes contained GS, 0.5 mM NHPPA and 40 µg/ml BSA in a final volume of 0.5 ml 50 mM KPi buffer (pH 7.4). Cytosolic incubations contained 2 mM NAD+ and 0.5 mM NHPPA in a final volume of 0.5 ml pyrophosphate buffer (pH 8.5). The reaction mixtures were placed in a water bath (37°C) and the reactions initiated by adding GS or NAD+. Control P450 or microsomal incubations omitted GS and control cytosolic incubations omitted NAD+. Percent recovery of NOPPA was determined by spiking either control reactions or reactions that were not incubated (0 time controls) with a known quantity of NOPPA. Recovery was always between 70 and 85%. After the appropriate time, the incubations were stopped by adding ethyl acetate, extracted three times with ethyl acetate, the extracts analyzed by TLC and the NOPPA quantitated as described above.


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In vitro formation of m7Gua as a result of metabolism of NOPPA
The metabolism of NOPPA resulting in the formation of m7Gua in calf thymus DNA was investigated using in vitro incubations containing various P450s or microsomal fractions to determine their ability to metabolize this compound to a methylating species. M7Gua was chosen as the marker for the formation of a methylating species since it is the most abundant DNA base modified by the methylating agents that are proposed to be formed from nitrosamines and it is readily amenable to analysis by HPLC using electrochemical detection (10,36).

In a previous study by Leung and Archer (26) it was shown that microsomes from NT rats metabolized NOPPA to a DNA methylating species, as determined by m7Gua formation, in vitro. When they used microsomes from rats pre-treated with phenobarital (PB), primarily an inducer 2B1/2, formation of m7Gua increased ~3-fold. In our assays, PB microsomes also readily metabolized NOPPA to a DNA methylating species (Table IGo) and our results with the PB microsomes are comparable with those of Leung and Archer (26). We also found that microsomes from rats pretreated with Pyr, primarily an inducer of 2E1, could metabolize NOPPA to a DNA methylating species and the amount of m7Gua formed was similar in the Pyr and PB microsomal assays. Purified rat 2B1 and rabbit 2E1 also readily catalyzed the formation of m7Gua confirming the ability of these P450s to metabolize this compound to a methylating species.


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Table I. Metabolism of NOPPA to form 7-methylguanine in vitro
 
In vitro formation of m7Gua as a result of metabolism of NHPPA
Rat and human microsomes also metabolized NHPPA to a DNA methylating species in vitro (Table IIGo), presumably by oxidation of NHPPA to NOPPA followed by P450-mediated {alpha}-hydroxylation as described above. Due to the low product yields and long incubation time, a relatively high concentration of microsomes was necessary to detect the methylated product in these assays. The high protein concentrations, particularly those used in the human and NT rat microsomal incubations, may be somewhat inhibitory and product formation may not have been optimal in some or all of the reactions. Therefore the data must only be viewed as a minimal estimate of activity. Approximately the same amount of m7Gua was formed in the incubations with human and NT rat microsomes (Table IIGo). Pretreatment of rats with PB or Pyr resulted in increased product formation of ~3–5-fold. No m7Gua could be detected when NHPPA was incubated with purified rat 2B1 in the reconstituted system. Since we have demonstrated that 2B1 can oxidize NHPPA to NOPPA (see below), it is speculated that detectable levels of product were not formed because the purified–reconstituted P450 system is unstable over the time period tested. We have found that product formation was only linear up to about 10 min during metabolic studies with NDPA and purified–reconstituted 2E1 (data not shown) suggesting that assays with the reconstituted systems may be limited to short time periods due to stability problems.


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Table II. Metabolism of NHPPA to form 7-methylguanine in vitro
 
Oxidation of NHPPA to NOPPA in vitro
The identity of the enzymes catalyzing the oxidation of NHPPA to NOPPA was investigated using TLC/densitometry to measure NOPPA formation from NHPPA in extracts of reaction mixtures containing purified P450s, rat microsomes or rat cytosol. Figure 2Go shows a representative TLC plate for the separation of the nitrosamines from the extract of an incubation containing NT rat microsomes. Under these conditions, NDPA could be separated from its oxidized derivatives as well as from NMPA (see standards in Figure 2Go). NHPPA was also separated into its geometric isomers, seen as doublets in the corresponding row. NMPA and NDPA could not be quantitated accurately by this method due to their volatility, resulting in significant losses from the extracts during drying with nitrogen.



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Fig. 2. Digital image of a thin layer chromatography plate used to separate NHPPA, NOPPA, NMPA and NDPA. Lanes 1–5 contained nitrosamine standards of 62.5, 125, 250, 375 and 500 ng, respectively. Lanes 6 and 7, extracts of reaction mixtures containing NT rat microsomes (0.25 mg/ml), 0.5 mM NHPPA and the NADPH-generating system processed as described in Materials and methods. Lane 8, extract of a reaction mixture containing NT rat microsomes (0.5 mg/ml) and 0.5 mM NHPPA without the NADPH-generating system processed as described in Materials and methods.

 
Incubation of 0.5 mM NHPPA with purified rat 2B1 and rabbit 2E1 in the reconstituted system resulted in the formation of 16.5 ± 3.1 and 20.0 ± 4.4 pmol NOPPA/pmol P450, respectively, in 1 h (Table IIIGo). For these reactions, linearity with time and protein concentrations was not determined. Therefore, the values cannot be considered rates, although they do demonstrate the ability of these P450s to readily catalyze this oxidation. However, rates of metabolism by microsomes and cytosol from NT rats were determined under conditions in which product formation was linear with time and protein concentration. At a 0.5 mM substrate concentration, the microsomes catalyzed NHPPA oxidation at a rate of 95.6 ± 16.5 pmol NOPPA/min/mg protein while cytosol catalyzed the reaction at a rate of 13.7 ± 3.0 pmol NOPPA/min/mg protein.


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Table III. Oxidation of NHPPA to NOPPA in vitroa
 
Oxidation of NHPPA to NOPPA in situ
Transfected and control Chinese hamster V79 cells and T5 cells, derived from lung tissue and T-antigen immortalized human liver epithelial cells, respectively, were investigated to determine their ability to oxidize NHPPA to NOPPA. T-Antigen immortalized human liver epithelial cells have been reported to express many phase II enzymes and have minimal, but detectable, P450-dependent activities (40). T5-2E1 cells transfected with human P450 2E1 were reported to have 2E1 catalytic activities comparable with primary hepatocytes from human liver (40). T5-neo cells contain the expression vector without the 2E1 insert. At 0.5 mM NHPPA, the amount of NOPPA formed in 23 h by the T5-2E1 cells was about 15-fold that formed by the T5-neo (Table IVGo). When the substrate concentration was lowered to 0.1 mM both T5-2E1 and T5-neo cells could still catalyze the ß-oxidation of NHPPA, although the amount of NOPPA formed in the T5-neo cells was too low to quantitate.


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Table IV. Oxidation of NHPPA to NOPPA by T5 and V79 cells in culturea
 
V79 cells transfected with rat 2B1 oxidized NHPPA to NOPPA (Table IVGo). Control V79 cells, which completely lack P450-dependent enzyme activities (41), also exhibited the ability to catalyze the formation of NOPPA at about one fifteenth of the amount formed in the V79 2B1 cells.

DNA methylation and toxicity in 2E1-expressing T5 cells by various nitrosamines
Methylation of DNA and toxicity by NDPA and its oxidized derivatives in T5-2E1 cells was investigated to assess the potential of the proposed oxidations to occur in a system that more closely resembles in vivo conditions (Figure 3Go; Table VGo). Cells dividing in culture are commonly very sensitive to alkylating agents (10,42,43). The potent animal carcinogen nitrosodimethylamine (NDMA) is activated by 2E1 to a methylating species (reviewed in 45) and was used as a positive control. NDMA and NDPA caused a dose dependent decrease in viability that was statistically significant at all concentrations tested (P < 0.05 by the Student's t-test), although NDPA was not as potent as NDMA (Figure 3Go). At a concentration of 50 µM, NDMA exposure resulted in the formation of 1.91 nmol m7Gua/mg DNA in 12 h while no m7Gua was detected after exposure to 1 mM NDPA for 12 h. NDPA is metabolized by 2E1 to a propylating species (8,30) and we attribute toxicity by this compound in our cells primarily to propylation of cellular macromolecules. NOPPA, the proposed proximate methylating nitrosamine intermediate of NDPA, also caused a dose-dependent decrease in viability. Although a statistically significant decrease was only observed at concentrations over 0.1 mM, at 2 mM NOPPA was equally as toxic as NDMA. At a concentration of 0.25 mM, NOPPA resulted in the formation of 0.95 nmol m7Gua/mg DNA in 12 h. Although we have shown that NHPPA is oxidized to NOPPA by these cells, NHPPA did not cause a decrease in viability at concentrations up to 2 mM, nor did it result in DNA methylation at a concentration of 1 mM. NMPA, the proximate methylating intermediate of NDPA proposed by Kruger, is N-demethylated and depropylated by 2E1 (23, unpublished observations). Exposure to this compound resulted in a dose dependent decrease in viability almost identical to that of NDMA and formed 2.02 ± 0.18 nmol m7Gua/mg DNA at a concentration of 50 µM after the 12 h incubation.



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Figure 3. Dose–response curves for nitrosamine cytotoxicity in T5 cells expressing human P450 2E1. NDPA ({circ}), NHPPA (x), NOPPA ({blacktriangleup}), NDMA (•) or NMPA ({square}) was added to the culture medium at the concentrations indicated and the relative survivals were determined as described in Materials and methods. Data are the mean ± SD from a representative experiment with each point determined in triplicate.

 

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Table V. Formation of 7-methylguanine in T5-2E1 cells exposed to various nitrosamines
 
It is interesting to note that the extent of methylation at the concentrations tested by each methylating nitrosamine correlates with the percent decrease in viability, or estimated decrease in viability for NOPPA, at those concentrations (Figure 3Go). This suggests that under the conditions tested, toxicity for these cells may be primarily due to the extent of macromolecule methylation. T5-neo cells exhibited no toxicity or m7Gua formation with any of the nitrosamines at concentrations up to 4 mM.


    Discussion
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 References
 
NDPA is metabolized to a methylating species in the rat liver (9), presumably through a sequential ß-oxidation to NOPPA, which is {alpha}-hydroxylated on the propyl chain and subsequently decomposes to yield a methylating species (Figure 1Go). Little is known about the enzyme(s) involved in the oxidation of NHPPA, the first intermediate in the pathway, to NOPPA. The principle objective of our investigation was to identify these enzymes. Additionally, the findings of Leung and Archer (26) identifying the enzymes involved in the oxidation of NOPPA to a DNA methylating species were confirmed and extended.

Leung and Archer (26) previously reported that liver microsomes from NT rats metabolized NOPPA to a DNA methylating species, as measured by the formation of m7Gua, and that product formation increased almost 3-fold when microsomes from PB treated rats were used, while no increase was observed when microsomes from 3-methylcholanthrene (a 1A1/2 inducer) treated rats were used. Our results with microsomes from rats pretreated with PB agree with those of Leung and Archer (26) and we additionally have found that microsomes from rats pretreated with Pyr also metabolize NOPPA to a DNA methylating species, suggesting the involvement of 2B1/2 and 2E1. The ability of rat 2B1 and rabbit 2E1 to catalyze the formation of a methylating species from NOPPA was confirmed with the purified isozymes. 2E1 is highly conserved across species and rabbit 2E1 exhibits a very similar substrate specificity to the rat and human orthologues (45,46). Therefore results with the rabbit isoform are expected to closely reflect the results that would be observed with the rat and human isoforms. Since rabbit 2E1 was readily available in our laboratory, it was used instead of the rat and human isoforms. 2E1 and 2B1/2 are constitutively expressed in the rat liver, although the levels of the latter is relatively low (reviewed in 47,48 and references therein). Based on our results as well as those of Leung and Archer (26), 2E1 and 2B1/2, as well as possibly unidentified isozymes of P450 other than 1A1/2 are likely contributing to NOPPA {alpha}-hydroxylation at a substrate concentration of 1 mM in the NT rat microsomes.

DNA methylation by NOPPA in the T5-2E1 cells demonstrates the ability of human 2E1 to activate this compound. The absence of a significant decrease in the viability of the T5-2E1 cells at lower concentrations of NOPPA (0.1 and 0.05 mM) does not necessarily indicate an inability of 2E1 to metabolize this compound at these concentrations. Competition for metabolism by other pathways or non-specific binding of NOPPA by cellular macromolecules may be effectively decreasing its intracellular concentration and metabolism by 2E1. NOPPA has been shown to be reduced to NHPPA in vivo and by rat liver cytosolic and microsomal fractions (18,49). Also, THLE cells may not be a sensitive indicator of toxicity due to robust DNA repair or other protective mechanisms. These studies are beneficial in initially defining the role of P450s in NOPPA {alpha}-hydroxylation; however, further studies are needed to identify the rat and human hepatic P450s that may contribute significantly to this reaction at concentrations expected to occur in vivo after environmental exposure to NDPA.

The ability of rat liver microsomes to metabolize NHPPA to DNA methylating species verifies the presence of microsomal enzyme(s) that are capable of catalyzing consecutive oxidations at rates sufficient to generate significant levels of the methylating species. It has previously been shown that microsomes from PB pre-treated rats could oxidize NHPPA to NOPPA (22). Based on our observations that NT microsomes are capable of oxidizing NHPPA to NOPPA, we presume that the sequential oxidation proposed in Figure 1Go is responsible for the generation of the methylating species. Deacetylation of NHPPA to NMPA, which is readily activated to a methylating species by microsomes, however, cannot be ruled out. Detection of m7Gua in the human microsomal sample at levels similar to those determined in the control rat microsomes, after incubation with NHPPA, suggests that this sequential oxidation pathway may also be relevant in humans.

Cytochrome P450s have been shown to oxidize secondary alcohols to carbonyl compounds (50). Incubations of NHPPA with purified rat 2B1 and rabbit 2E1 in the reconstituted system conclusively demonstrated for the first time that P450s can catalyze the oxidation of this secondary alcohol. Microsomes from NT rats also catalyzed this oxidation. Since the microsomal oxidation was dependent on the NADPH-generating system, conditions in which NADP+ concentrations are kept to a minimum, we presume that it is mediated by P450s. Rat cytosol also oxidized NHPPA in the presence of NAD+, suggesting that cytosolic alcohol dehydrogenases have a capacity to catalyze this reaction. The activity in the cytosol was about one seventh of that in the microsomes, per milligram of protein. Estimating that the cytosolic protein is about 2.5 times the amount of microsomal protein in the mammalian liver (51), our in vitro results suggest that the P450(s) will be significant, if not the primary, contributors to NHPPA ß-oxidation at a 0.5 mM substrate concentration.

In vitro conditions may vary greatly from those in vivo and oxidative pathways other than those examined in vitro may contribute to NHPPA ß-oxidation. To help address these concerns, ß-oxidation of NHPPA in cell cultures was examined. Formation of NOPPA from NHPPA in V79 control cells lacking P450s confirms the ability of pathways other than those utilizing P450s to mediate this reaction. However, as can be seen in Table IVGo, the amount of NOPPA formed in the T5-2E1 and V79 2B1 P450 cell lines was approximately 15-fold the amount formed in the respective control cell lines. This indicates that P450s are the primary contributors to NHPPA ß-oxidation in these cell lines and lends support to the supposition that this may also be the case in vivo, although several other factors must be considered when interpreting these results. First, as a result of the catalytic cycle of P450, which requires the reduction of molecular oxygen, reactive oxygen species (ROS) are generated as by-products in the presence or absence of substrates (52) and the presence of P450 may alter the redox status of the cells. ROS and perturbations in cellular redox status are well known to have inductive and suppressive effects on a large number of genes (reviewed in 53,54). It is possible that the levels of enzymes other than P450 which could be involved in the oxidation of NHPPA may have increased in the P450 transfected cells, resulting in an overestimation of the contribution of P450s. Second, the cell lines, which are derived from the human liver epithelium and hamster lung, may not be representative of target cells and may differ from the actual target cells in vivo with respect to pathways critical for NHPPA oxidation. Third, NHPPA is an intermediate in the sequential oxidation pathway of NDPA and the concentrations used here may be well above those formed in vivo. Therefore, the relative contribution of the various enzymes involved in NHPPA oxidation may change when the levels of NHPPA are decreased to concentrations expected to occur in vivo after environmental exposure to NDPA.

Despite the abilities of T5-2E1 cells to oxidize NHPPA to NOPPA and microsomes to metabolize NHPPA to a methylating species, no toxicity or DNA methylation was detected as a result of exposure of the T5-2E1 cells to NHPPA. One reasonable explanation for this is the generated NOPPA may be extensively diluted into the large volume of media used in these assays. Based on the amount of NOPPA formed from 0.5 mM NHPPA in the T5-2E1 cells and on the cell culture conditions, the concentration of NOPPA in the cultures after 23 h is estimated to be approximately 12 µM. This is well below the concentration necessary to elicit a decrease in viability in this assay. Therefore, deacetylation of NHPPA to NMPA, which was shown to be a potent toxin and methylating species in these cells, cannot be ruled out as the methylating species because the generated NMPA would also be expected to be extensively diluted. With this rationale, it can also be argued that methylation by NOPPA in the cells is not necessitated by an initial deacetylation step to NMPA, but is due to a single enzymatic reaction, {alpha}-hydroxylation of NOPPA. As proposed by Leung and Archer (13), {alpha}-hydroxylation of NOPPA on the propyl chain is believed to result in the formation of the oxopropyldiazotate which rapidly breaks down to the methylating species.

The results of this study corroborate previous studies, suggesting that the metabolism of NDPA to a methylating species through the sequential oxidation pathway may be relevant in the rat liver. We have shown that P450s can oxidize NHPPA to NOPPA, and our data suggest that P450s may play a critical role in the reactions by which NHPPA is oxidized to NOPPA at the concentrations tested. Due to the similarities in the structures of the dialkylnitrosamines and the overlapping substrate specificities of the P450s, it is likely that P450s 2E1 and 2B1 will oxidize secondary alcohol functions on other long chain nitrosamine carcinogens as well. Cytosolic NAD+-dependent alcohol dehydrogenases appear to be able to catalyze this reaction as well; however, involvement of additional oxidative pathways cannot been ruled out. Our results, together with those of Bellec et al. (21), who have shown that human 2E1 can ß-hydroxylate NDPA, indicate that 2E1 can catalyze all three oxidations of NDPA required to form the methylating species. We have previously found 2E1 to be the predominant human liver isoform responsible for the {alpha}-hydroxylation of NDPA, which results in the propylating species, at low micromolar concentrations (30). 2E1 is constitutively expressed and highly inducible by alcohol (55) suggesting that this isozyme may play a critical role in the hepatic activation of NDPA to both a propylating and a methylating species under some conditions. Additional studies are needed to clearly define the roles of the identified enzymes, and potentially other enzymes, in the metabolism of NDPA to a methylating species.

In this study we focused on P450s 2E1 and 2B1 which were previously shown to be the primary contributors to NDPA {alpha}-hydroxylation in rat liver microsomes (8,20). Here we show that these isozymes can also catalyze oxidations of oxidized derivatives of NDPA. This suggests that the substrate specificity for oxidation, at least by 2E1 and 2B1, may be primarily directed by the size of the alkyl chains and not by the nature of the functional groups within the molecule. Further studies are needed with additional NDPA derivatives and P450 isozymes to support this generalization.


    Notes
 
4 To whom correspondence should be addressed at Department of Pharmacology, 2301 Medical Sciences Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632, USA E-mail: phollen{at}umich.edu Back


    Acknowledgments
 
We thank Dr P.Watkins for the human liver sample, Drs J.Doehmer and T.Kocarek for the V79 cells and Dr A.Abe for help with the digital imaging of the TLC plates. 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 August 30, 2000; revised November 27, 2000; accepted December 5, 2000.





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