Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase

Hirotaka Kushida, Ken-ichi Fujita, Akihiro Suzuki, Masami Yamada1, Toru Endo2, Takehiko Nohmi1 and Tetsuya Kamataki3

Laboratory of Drug Metabolism, Division of Pharmacobio-dynamics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812,
1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku Tokyo 158-8501 and
2 Central Research Laboratories, Tsumura & Co., Inashiki-gun, Ibaraki 300-1192, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established. The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of Salmonella. Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR. Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined. CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN. CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK. The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine. In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).

Abbreviations: {delta}-ALA, {delta}-aminolevulinic acid; G-6-P, glucose 6-phosphate; G-6-PDH, glucose 6-phosphate dehydrogenase; IPTG, isopropyl ß-D(–)-thiogalactopyranoside; MC, minimal concentration; NDAA, N-nitrosodiamylamine; NDBA, N-nitrosodibutylamine; NDEA, N-nitrosodiethylamine; NDMA, N-nitrosodimethylamine; NDPA, N-nitrosodipropylamine; NMPhA, N-nitrosomethylphenylamine; NNK, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone; NNN, N-nitrosonornicotine; NPYR, N-nitrosopyrrolidine; OR, NADPH-cytochrome P450 reductase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-Alkylnitrosamines are recognized as one of the most potent chemical carcinogens present widely in the environment, as evidenced by the fact that they induce cancer in various organs in experimental animals, and maybe even in humans (15). The N-alkylnitrosamines require metabolic activation to exert their genotoxicity. The first activation step of N-alkylnitrosamines is thought to be hydroxylation of the carbon atom located at the {alpha} position of the N-nitroso group. The reaction is mainly catalyzed by cytochrome P450 (2,5). The subfamilies of cytochromes P450 mainly involved in hydroxylation of N-nitrosamines are thought to be CYP2E and CYP2A in humans (68).

The CYP2E subfamily contains two forms, CYP2E1 and CYP2E2 (911). Human CYP2E1 metabolizes drugs such as chlorzoxazone (12) and is responsible for the metabolic activation of N-nitrosodialkylamines such as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) (6). Recently, the metabolism of N-nitrosodialkylamines such as NDMA, NDEA, N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosodiamylamine (NDAA) was investigated using human liver microsomes and cDNA-expressed human cytochromes P450 (13). The results showed that NDMA was metabolized primarily by human CYP2E1.

The CYP2A subfamily consists of at least 12 distinct forms (14). The forms in the CYP2A subfamily efficiently catalyze coumarin 7-hydroxylation (1518). In humans, CYP2A6 is one member of the CYP2A subfamily. CYP2A6 is responsible for the metabolic activation of N-alkylnitrosamines including NDMA, NDEA, N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine (6,19). Recently, we successfully established a Salmonella typhimurium strain YG7108 2A6/OR co-expressing CYP2A6 and human NADPH-cytochrome P450 reductase (OR). The mutagen-activating capacity of CYP2A6 for various N-alkylnitrosamines was determined with genetically engineered bacterial cells. It was demonstrated that CYP2A6 was responsible for the metabolic activation of N-nitrosomethylphenylamine (NMPhA). To our knowledge, mutagenicity of NMPhA could not be detected by the Ames test using a parental YG7108 strain in the presence of S9 (9000 g supernatant fraction of liver homogenates) prepared from rat liver.

Despite these studies, no comparative studies have been reported so far to clarify the roles of human CYP2E1 and CYP2A6 in the metabolic activation of a series of N-alkylnitrosamines. Thus, in the present study, we first established a new S.typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 and human OR. We report herein a comparison of the capacity of human CYP2E1 with that of CYP2A6 to metabolically activate eight alkyl derivatives of N-nitrosamines using the YG7108 2E1/OR and YG7108 2A6/OR strains of Salmonella.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
NDMA, NDEA, isopropyl ß-D(–)-thiogalactopyranoside (IPTG), 4-nitrophenol, coumarin and cytochrome c were purchased from Wako Pure Chemical Industries (Osaka, Japan). NNN and NNK were obtained from Toronto Research Chemicals (Toronto, Canada). Glucose 6-phosphate (G-6-P), glucose 6-phosphate dehydrogenase (G-6-PDH) and NADP+ were from Oriental Yeast (Tokyo, Japan). NDPA, NDBA, NMPhA, N-nitrosopyrrolidine (NPYR) and {delta}-aminolevulinic acid ({delta}-ALA) were from Tokyo Chemical Industry (Tokyo, Japan).

Salmonella typhimurium tester strains and plasmids
Salmonella typhimurium tester strains and plasmids used in this study are summarized in Table IGo. YG7108, derived from TA1535, lacks two O6-methylguanine-DNA methyltransferase genes, ada and ogt (20). An expression plasmid pCYP2E1/OR (Figure 1Go) carrying CYP2E1 and human OR cDNAs (21) was first modified by introduction into S.typhimurium strain LB5000 (RM+) (22) and then the plasmid was introduced into YG7108 cells.


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Table I. Salmonella typhimurium strains and plasmids established or used in the present studya
 


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Fig. 1. Structure of a plasmid to co-express cytochrome P450 and OR in S.typhimurium YG7108 cells. The 9.3 kb plasmid contains cDNAs for cytochrome P450 and OR, which are linked to the tac promoter and terminator, respectively.

 
Culture conditions for expression of CYP2E1 or CYP2A6 with OR in S.typhimurium YG7108
Expression of CYP2E1 or CYP2A6 with OR in the Salmonella cells was achieved as follows. Twenty microliters of a bacterial stock solution were inoculated into 10 ml of Luria–Bertani medium supplemented with ampicillin (100 mg/ml), kanamycin (25 mg/ml) and chloramphenicol (10 mg/ml). The bacteria were grown with shaking at 37°C for 12 h. Two milliliters of the culture were inoculated into 200 ml of a modified Terrific Broth (23) and the bacteria were grown with shaking at 37°C for 2 h prior to induction with 1.5 mM IPTG. The expression of recombinant proteins was achieved by a further incubation at 30°C for 18 h with shaking.

Assay of catalytic activity
All assays were carried out with whole bacterial cells expressing CYP2E1 or CYP2A6 with OR. The amount of the bacterial preparation added to the incubation mixture will be shown as the amount of cytochrome P450. All reactions were initiated by addition of substrate.

4-Nitrophenol hydroxylase activity of CYP2E1 expressed in the bacterial cells was assayed essentially as described by Tassaneeyakul et al. (25). Briefly, the incubation mixture contained 100 mM potassium phosphate buffer (pH 6.8), 200 µM 4-nitrophenol and 3.3 pmol of CYP2E1 in a final volume of 0.5 ml. Incubations were carried out at 37°C for 60 min. The metabolite was extracted with 4 ml of diethyl ether. Analysis of the metabolite, 4-nitrocatechol, was performed by HPLC (Shimadzu, Kyoto, Japan) on a Capcell Pak C18 column (4.6x250 nm, SG120A, 5 µm; Shiseido, Tokyo, Japan) with a SPD-6A UV absorbance detector (Shimadzu, Kyoto, Japan). The column temperature was 40°C. Metabolites were separated using a solvent system containing 22% acetonitrile, 1% acetic acid and 77% trimethylamine (30 mM, pH 3.0). The flow rate was 1.0 ml/min. The elution of 4-nitrophenol and its metabolites was monitored at 250 nm.

Coumarin 7-hydroxylase activity of CYP2A6 expressed in whole bacterial cells was measured essentially according to the method of Pearce et al. (24). Briefly, the incubation mixture consisted of 25 mM potassium phosphate buffer (pH 7.4), 50 µM coumarin and 10 pmol of CYP2A6 in a final volume of 1.0 ml. Incubations were carried out at 37°C for 15 min. The metabolite, 7-hydroxycoumarin, was determined fluorometrically.

Mutation assay
Mutation assays with YG7108 2E1/OR and YG7108 2A6/OR were carried out as follows. Cultures were in 200 ml of modified Terrific Broth (23) and were grown with shaking at 37°C for 2 h prior to induction with 1.5 mM IPTG. Expression of recombinant proteins was achieved by a further incubation at 30°C for 18 h with shaking. The number of cells was adjusted to give 1–2x109 cells/ml by dilution with modified Terrific Broth. The assay was carried out as described by Maron and Ames (26) with minor modifications. Bacterial cells were pre-exposed to an N-alkylnitrosamine at 37°C for 20 min before plating.

The plates were incubated at 37°C for 2 days. Assays were carried out in triplicate at each dose. The results were judged as positive when the number of colonies increased in a dose-dependent manner and reached a level twice as high as that obtained with vehicle alone as a control. The minimal concentration (MC) value of N-alkylnitrosamines was defined as the concentration of a promutagen giving a positive result.

Other methods
The content of cytochrome P450 in bacterial cells was determined by Fe2+–CO versus Fe2+ difference spectra, according to the method of Omura and Sato (27). Salmonella cells were suspended in 100 mM potassium phosphate buffer (pH 7.4) containing 20% (v/v) glycerol and 0.2% (w/v) Emulgen 911. The difference spectra were recorded using a UV-visible spectrophotometer model MPS-2000 (Shimadzu, Kyoto, Japan). The OR activity in sonicated bacterial cells was measured with cytochrome c as the electron acceptor by detecting the absorbance change at 550 nm at 20°C according to the method of Phillips and Langdon (28). Protein concentrations were determined as described by Lowry et al. (29).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of CYP2E1 or CYP2A6 together with OR in S.typhimurium YG7108
An expression plasmid pCYP2E1/OR carrying CYP2E1 cDNA together with OR cDNA was introduced into S.typhimurium YG7108, which shows an ada- and ogt-deficient genotype. The expression of CYP2E1 holoprotein in YG7108 2E1/OR was determined with Fe2+–CO versus Fe2+ difference spectra (Figure 2Go). The expression level of CYP2E1 in comparison with that of CYP2A6 and the level of OR in bacterial cells are summarized in Table IIGo. The expression levels of CYP2E1 and CYP2A6 were 15 ± 3 and 77 ± 8 nmol/l culture, respectively. No detectable cytochrome P450 was seen in the YG7108 pCW cells.



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Fig. 2. Typical CO difference spectra of CYP2E1 and CYP2A6 expressed in established S.typhimurium. The content of CYP2E1 or CYP2A6 in S.typhimurium cells was determined by Fe2+–CO versus Fe2+ difference spectra.

 

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Table II. Expression levels of cytochrome P450 and OR in StyphimuriumYG7108 cells co-expressing CYP2A6 or CYP2E1 and OR
 
The expression levels of OR were 390 ± 17 and 470 ± 20 µmol cytochrome c reduced/l culture for YG7108 2E1/OR and YG7108 2A6/OR, respectively. Only a small amount of OR activity was detectable in strain YG7108 pCW.

Enzyme activities in established S.typhimurium whole cells
The catalytic activities of CYP2E1 and CYP2A6 expressed in YG7108 2E1/OR and YG7108 2A6/OR cells were determined using as substrates 4-nitrophenol for CYP2E1 and coumarin for CYP2A6 (Table IIIGo). The catalytic activities of CYP2E1 and CYP2A6 for 4-nitrophenol hydroxylation and coumarin 7-hydroxylation were 19.5 ± 2.2 and 16.3 ± 1.8 nmol/min/nmol cytochrome P450, respectively. Activity was not detectable in the parental YG7108 pCW cells.


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Table III. Catalytic activities of CYP2E1 and CYP2A6 in S.typhimurium YG7108 cells
 
Mutation assay with YG7108 2E1/OR and YG7108 2A6/OR cells for N-alkylnitrosamines
The mutagen-producing activity of CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using established S.typhimurium YG7108 2E1/OR and YG7108 2A6/OR, respectively. Eight N-alkylnitrosamines, NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNK and NNK, were employed as promutagens. The results are shown in Figure 3Go. CYP2E1 expressed in the genetically engineered S.typhimurium cells activated NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN, while CYP2A6 was capable of activating all promutagens tested. The MC values defined as described in Materials and methods for YG7108 2E1/OR ranged from 1.43 µM for NDPA to 1.43 mM for NDMA. The MC values for YG7108 2A6/OR ranged from 0.143 µM for NMPhA to 1.43 mM for NDMA (Table IVGo).



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Fig. 3. Comparison of the mutagen-activating capacity of CYP2E1 and CYP2A6 for various N-alkylnitrosamines. Salmonella typhimurium expressing CYP2E1 or CYP2A6 was treated with NDMA (A), NDEA (B), NDPA (C), NDBA (D), NMPhA (E), NPYR (F), NNK (G) and NNN (H) at 37°C for 20 min. Immediately after preincubation, the reaction mixture was poured onto a minimal glucose plate with top agar. The plates were incubated at 37°C for 2 days before the number of His+ revertants per plate was counted. Data points are shown as the mean (n = 3). Bars indicate standard deviation.

 

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Table IV. Comparison of the MC values of CYP2E1 with those of CYP2A6 with different N-alkylnitrosamines
 
The capacity of CYP2E1 and CYP2A6 to activate N-alkylnitrosamines is summarized in Table VGo. CYP2E1 activated N-alkylnitrosamines with a capacity ranging from 12.2 to 476 induced revertants/min/pmol cytochrome P450/nmol mutagen for NNK and NDEA, respectively. CYP2A6 activated N-alkylnitrosamines with a capacity ranging from 0.322 to 7590 induced revertants/min/pmol cytochrome P450/nmol mutagen for NDMA and NMPhA, respectively. The ratios of the values with CYP2A6 to those with CYP2E1 are also shown in Table VGo. In the case of NDMA, NPYR and NDEA, the ratio was below 1.0, indicating that CYP2E1 activates these promutagens more efficiently than does CYP2A6. On the other hand, the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. These results indicate that CYP2E1 is mainly responsible for the metabolic activation of N-alkylnitrosamines with relatively short alkyl chain(s), whereas CYP2A6 is predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).


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Table V. Comparison of the mutagen-activating capacity of CYP2E1 with that of CYP2A6 with N-alkylnitrosamines
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-alkylnitrosamines cause cancers in many organs of experimental animals and are considered as one of the most important chemical carcinogens in the environment (15). The metabolic activation of N-alkylnitrosamines by cytochrome P450 enzymes may be critical in the initiation of human cancer (32). CYP2E1 and CYP2A6 expressed in human liver microsomes have been reported to be the major enzymes in the metabolic activation of N-alkylnitrosamines such as NDMA, NDEA, NNK and NNN (68).

To clarify the roles of CYP2E1 and CYP2A6 in the metabolic activation of N-alkylnitrosamines in detail, we established Salmonella YG7108 cells expressing CYP2E1 or CYP2A6 together with OR (YG7108 2E1/OR and YG7108 2A6/OR). CYP2E1 and CYP2A6 were sufficiently expressed in the genetically engineered Salmonella cells. In the case of CYP2E1, the level of CYP2E1 expressed in 1 l of the culture of YG7108 2E1/OR cells corresponded to that expressed in 23 g of human liver, assuming that 1 g of human adult liver contained ~0.66 nmol of CYP2E6 protein, referring to the data reported by Shimada et al. (30). The expression level of CYP2A6 in 1 l of a culture of YG7108 2A6/OR cells corresponded to that expressed in 193 g of human liver, assuming that 1 g of human adult liver contained ~0.4 nmol of CYP2A6 protein.

The endogenous electron transport system of Salmonella YG7108 cells is able to support only a small portion of the cytochrome P450 activity (33). Thus, OR, which is capable of transferring electrons from NADPH to cytochrome P450, was co-expressed in the Salmonella cells with cytochrome P450 in order to obtain full catalytic activity of cytochrome P450. As a result, CYP2E1 and CYP2A6 expressed in the Salmonella cells efficiently catalyzed 4-nitrophenol hydroxylation and coumarin 7-hydroxylation, respectively (Table IIIGo).

Reactive intermediates produced by cytochrome P450 are, in general, believed to be chemically unstable and reactive with bacterial cell components. Therefore, as is the case with the classical Ames test, the reactive metabolites are assumed to bind to components on the cell surface if the reactive intermediates are formed outside the cell by drug-metabolizing enzymes contained in S9 mix. Expression of cytochrome P450 in the Salmonella cells is expected to lead to intracellular production of reactive intermediates, resulting in efficient binding of reactive metabolites to DNA, causing mutations in the Salmonella genome. YG7108 cells lack two O6-methylguanine-DNA methyltransferase genes, ada and ogt. Since N-alkylnitrosamines induce DNA mutations via alkylation of purines and pyrimidines (34), the properties of the established Salmonella strain might also make it highly sensitive to N-alkylnitrosamines.

The capacity for mutagenic activation of N-alkylnitrosamines was not affected by the addition of NADPH, suggesting that Salmonella could generate sufficient NADPH for cytochrome P450 activity.

We have shown that the contribution of CYP2E1 was larger than that of CYP2A6 in the activation of NDEA. However, previous reports demonstrated a high correlation between coumarin 7-hydroxylase activity and the formation of acetaldehyde from NDEA and a poor correlation between CYP2E1 content and acetaldehyde formation from NDEA (13,35). Thus, the previous data indicated that CYP2A6 was involved in the metabolic activation of NDEA, while CYP2E1 was not. The apparent discrepancy between the previous and the present data may be accounted for by the fact that the previous investigators measured acetaldehyde formation from NDEA at high concentrations of NDEA (2.00–4.00 mM), because of the low sensitivity of the method (36) to detect aldehyde formation, while we carried out experiments with NDEA at a low concentration (7.14 µM).

Bellec et al. (13) examined aldehyde formation via {alpha}-hydroxylation using symmetrical N-dialkylnitrosamines such as NDMA, NDEA, NDPA, NDBA and NDAA. They demonstrated that CYP2E1 was responsible for {alpha}-hydroxylation of N-alkylnitrosamines with short alkyl chains, measuring aldehyde formation from the N-alkylnitrosamines. The same tendency was found in the present study on the role of CYP2E1 in the mutagenic activation of these chemicals as that seen in the previous study. In the present study we also examined the role of CYP2E1 in the metabolic activation of N-alkylnitrosamines with non-symmetrical alkyl chains, such as NNK and NMPhA, in addition to N-alkylnitrosamines with symmetrical alkyl chains. Although NNK and NMPhA possess a methyl group at the {alpha} position of the carbon atom bonded to the nitroso group, these were not efficiently activated by CYP2E1. These results suggest that the methyl group at the {alpha} position of the carbon atom bonded to the nitroso group may not be a substrate determinant for CYP2E1 and that molecular mass may be the main determinant of a CYP2E1 substrate. On the other hand, we have demonstrated that CYP2A6 is involved in the metabolic activation of all the N-alkylnitrosamines examined, while CYP2E1 was not responsible for metabolic activation of NMPhA and NNN (Figure 3Go and Table VGo). The results suggest that CYP2A6 has a wider substrate specificity for N-alkylnitrosamines than does CYP2E1. The metabolic activation of many other N-alkylnitrosamines should be investigated to prove this hypothesis.


    Notes
 
3 To whom correspondence should be addressed Email: kamataki{at}pharm.hokudai.ac.jp Back


    Acknowledgments
 
We thank Prof. F.W.Dahlquist and Dr H.Satoh for supplying the pCW vector. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, by the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan and also by research funds from the Japanese Society of Alternatives to Animal Experiments.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 30, 1999; revised February 22, 2000; accepted February 24, 2000.