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Departments of Pharmacology and Toxicology (G.B.J.S., L.L.B., T.E.M.), Surgery (K.R.R., D.P.), and Medicine (T.E.M.), and School of Environmental Studies (T.E.M.), Queen's University, Kingston, Ontario, Canada; and Department of Pharmacology and Toxicology (J.R.B.), University of Western Ontario, London, Ontario, Canada
(Received March 3, 2003; accepted June 12, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-carbon hydroxylation endpoint metabolites)
ranged from 0.002100 to 0.005685% -hydroxylation/mg of protein/min.
Overall, production of bioactivation metabolites was greater than that of
detoxication (i.e., N-oxidation) products. Based on total
bioactivation, subjects could be classified as high or low NNK bioactivators.
In the presence of an NADPH-generating system, microsomal formation of the
endpoint metabolite 1-(3-pyridyl)-1-butanone-4-carboxylic acid (keto acid) was
consistently higher than that of all other -carbon hydroxylation
endpoint metabolites. Contributions of cytochrome P450 (P450) enzymes to NNK
oxidation were demonstrated by NADPH dependence, inhibition by carbon
monoxide, and inhibition by the nonselective P450 inhibitors proadifen
hydrochloride (SKF-525A) and 1-aminobenzotriazole (ABT), particularly in lung
microsomes from high bioactivators. At 5.0 mM, ABT inhibited total NNK
bioactivation by 54 to 100%, demonstrating the importance of ABT-sensitive
enzyme(s) in human pulmonary NNK bioactivation. Contributions of CYP2A6 and/or
CYP2A13, as well as CYP2B6, to NNK bioactivation were also suggested by
selective chemical and antibody inhibition in lung microsomes from some
subjects. It is likely that multiple P450 enzymes contribute to human
pulmonary microsomal NNK bioactivation, and that these contributions vary
between individuals.
). NNK and
its keto-reduced major metabolite
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) require metabolic
activation via -carbon hydroxylation to form unstable intermediates
that spontaneously decompose to produce alkylating agents. Reaction of these
alkylating metabolites with DNA is believed to initiate carcinogenicity
(Hecht, 1998)
(Fig. 1). Pyridine
N-oxidation of NNK and NNAL results in formation of excretable
N-oxides, which are detoxication products
(Hecht, 1998)
(Fig. 1).
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In rodents, cytochrome P450 (P450) enzymes play the major role in pulmonary
NNK bioactivation (reviewed in Hecht,
1998). The situation in human lung is more complex, with much
lower levels of NNK bioactivation compared with rodents, and evidence
implicating multiple enzyme systems including P450s, lipoxygenases (LOXs),
prostaglandin H synthases (PHSs), and other peroxidases (Smith et al.,
1992,
1995,
1999).
Our previous observation of inhibition of NNK bioactivation by SKF-525A in
human lung cells (Smith et al.,
1999), coupled with evidence of the abilities of various
recombinant human P450s to catalyze NNK bioactivation
(Hecht, 1998;
Kushida et al., 2000;
Su et al., 2000), implicated
cytochrome P450 in pulmonary NNK metabolism. Previous studies have
demonstrated P450-catalyzed NNK bioactivation in human whole lung microsomes.
However, information about roles of specific P450 enzymes was based on results
in lung microsomes from a single patient in one study
(Smith et al., 1992) and only
three individuals in another (Smith et
al., 1995). Our demonstration of considerable interindividual
variability in NNK metabolism in lung cells
(Smith et al., 1999), and
variable human lung metabolism of other carcinogens
(Harris et al., 1976;
De Flora et al., 1987),
indicated that valid conclusions regarding roles of different
biotransformation enzymes required further examination.
The objective of the present study was to assess the contributions of
various biotransformation enzymes pertinent to human pulmonary microsomal NNK
metabolism using antibody and chemical inhibitors. P450 enzymes to be examined
were selected based on previously demonstrated expression in human lung and by
metabolic activity toward NNK in expression systems (reviewed in
Hecht, 1998; and
Su et al., 2000). Thus,
CYP2A6, CYP2A13, CYP3A4, CYP3A5, CYP2E1, and CYP2B6 were considered to be P450
enzymes most likely to be active in the human pulmonary biotransformation of
NNK. Since our previous results suggested possible non-P450 contributions to
NNK biotransformation in freshly isolated alveolar macrophages
(Smith et al., 1999), and PHS
has been proposed as a potential NNK bioactivating enzyme (Rioux and
Castonguay, 2000,
2001), its involvement in
human lung microsomal NNK metabolism was also assessed.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-Methylbenzyl-1-aminobenzotriazole (MB) and
N-benzyl-1-aminobenzotriazole (BBT) were synthesized as previously
described (Mathews and Bend,
1986). All other chemicals were reagent grade or better and were
obtained from common commercial suppliers.
Tissue Procurement. Following informed consent, sections of
peripheral human lung were obtained during clinically indicated lobectomy as
described previously (Smith et al.,
1999). Tissue specimens were cut into 1.5-cm3 pieces,
wrapped in aluminum foil, frozen in liquid N2, and stored at
-80°C until microsome preparation. Tissues were examined histologically
for the presence of anomalies as described in
(Smith et al., 1999). Patients
were characterized with respect to age, gender, surgical diagnosis, possible
occupational exposure to carcinogens and/or biotransformation enzyme
inducers/inhibitors, drug treatment 1 month before surgery, and self-reported
smoking history. Patients were classed as former smokers if self-reported
smoking history indicated cessation greater than 2 months before surgery. This
time interval was chosen to eliminate the inductive effects of cigarette smoke
on biotransformation enzymes (McLemore et
al., 1990).
Preparation of Human Whole Lung Microsomes. Tissue sections wrapped
in foil were thawed on ice for 15 min. All subsequent manipulations were
carried out on ice or at 4°C. The lung tissue was rinsed, chopped, and
then homogenized using a Polytron homogenizer in 3 volumes of 0.1 M potassium
phosphate buffer containing 1.15% KCl (pH 7.4). Microsomes were prepared by
differential centrifugation (Donnelly et
al., 1996). Protein concentration was determined by the method of
Lowry et al. (1951), using
bovine serum albumin as a standard.
Incubations with NNK. [5-3H]NNK was purified to >99.8%
as described (Smith et al.,
1999) before use. Incubation mixtures contained 0.1 M potassium
phosphate buffer plus 1.15% KCl (pH 7.4), 4.2 µM [5-3H]NNK, 1.0
mM EDTA, 3.0 mM MgCl2, and 1.0 mg microsomal protein in a total
volume of 1.0 ml. The NADPH-generating system consisted of 5.0 mM glucose
6-phosphate, 2.0 units of glucose-6-phosphate dehydrogenase, and 1.25 mM NADP.
The reaction mixture was incubated for 15 min at 37°C in a Dubnoff
metabolic shaker. Reactions were terminated by addition of 300 µl of 25%
zinc sulfate and 300 µl of saturated barium hydroxide. Samples were
centrifuged at 2,500g for 5 min, and supernatants were frozen in
liquid N2 and stored at -80°C until analysis. Inhibitors were
dissolved in methanol, which constituted 1.0% (v/v) of the total incubation
volume; control incubations contained 1.0% methanol (v/v), which previously
has been shown to have no appreciable effect on human lung microsomal NNK
metabolism (Smith et al.,
1995). Isozyme nonselective inhibitors of P450, SKF-525A
(Ono et al., 1996) or ABT
(Mathews et al., 1985), were
used to assess overall P450 contribution to NNK metabolism. 8-MS (for CYP2A6;
Koenigs et al., 1997) and,
potentially, CYP2A13 (Su et al.,
2000); TAO (for CYP3A4/5;
Newton et al., 1995); and ORP,
MB, or BBT (for CYP2B6; Mathews and
Bend, 1986; Reidy et al.,
1989) were used to assess contributions of specific P450 enzymes.
PHS contributions were assessed using INDO
(Smith and DeWitt, 1995).
Effects of NDGA, a LOX inhibitor (Van der
Merwe et al., 1993), were also assessed. For immunoinhibition
studies, 1.0 mg of microsomal protein and 200 µg of either anti-CYP2A6,
CYP3A4/5, CYP2E1, or CYP2B6 supplied in 25 mM Tris buffer (pH 7.5) were
incubated on ice for 20 min before use in incubations, as recommended by the
supplier. Incubations in the absence of inhibitory antibodies also contained
the Tris buffer supplied with the antibodies. All incubations were performed
in duplicate, and components were preincubated for 15 min at 37°C to allow
for the mechanism-based inhibitors to exert their effects before addition of
[5-3H]NNK. Linearity of NNK metabolite formation by human lung
microsomes has been demonstrated for at least 30 min
(Smith et al., 1992). For
incubations with carbon monoxide (CO), mixtures were bubbled with CO for 1 min
immediately before addition of [5-3H]NNK. Due to limitations in
tissue availability, not all inhibitors could be tested in microsomes from all
patients.
Assessment of NNK Biotransformation. Microsomal NNK
biotransformation was analyzed as described
(Smith et al., 1999), except
that 1.0 ml of the terminated, filtered reaction mixture was injected onto the
high-performance liquid chromatography column, and radiometric quantitation of
eluted products was accomplished with a ß-ram radioactivity flow detector
(IN/US Systems Inc., Tampa, FL) with Uniscint BD as the scintillation cocktail
at a 3:1 cocktail/eluate flow mixture ratio. The metabolite reference
standards solution contained NNK and each of the seven metabolites dissolved
in methanol at 6.25 mM, of which 5.0 µl were injected onto the column. NNK
metabolite values from control incubates containing boiled microsomes were
subtracted from each individual patient's microsomal metabolite profile.
Microsomal incubations were analyzed in duplicate. For each NNK metabolite,
the amount produced was expressed as a percentage of the total radioactivity
recovered from [5-3H]NNK plus metabolites per milligram of protein
per minute, to account for any differences in recovery of [5-3H]NNK
from incubate to incubate. Metabolite peaks were quantitated only if they were
at least twice background radioactivity levels.
Data Analysis and Presentation. For microsomal NNK
biotransformation, individual metabolite values represent the mean of
duplicate incubations. When grouped, microsomal data are presented as means
± S.D. Statistically significant differences in individual microsomal
metabolite formation and differences following inhibitor treatments were
determined by repeated measures analysis of variance (ANOVA) followed by the
Tukey-Kramer test or Dunnett's multiple comparisons test, respectively. When
Bartlett's test revealed heterogeneity of variance, a positive integer
(k = 1) was added to each datum because some points were equal to
zero (nondetectable), and either log or reciprocal transformation was
performed on the resultant values prior to conducting ANOVA
(Zivin and Bartko, 1976). If
the transformed data did not achieve homogeneity of variance, the Freidman
nonparametric repeated measures test was used
(Zivin and Bartko, 1976).
P < 0.05 was considered statistically significant in all
cases.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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[5-3H]NNK Biotransformation in Whole Lung Microsomes.
Metabolism of [5-3H]NNK in human lung microsomes resulted in the
formation of hydroxy acid, keto acid, diol, keto alcohol,
NNAL-N-oxide, NNK-N-oxide, and NNAL. NNAL was the major
metabolite, and its formation ranged from 0.52 to 1.3%/mg of protein/min
(representing 
5–13 pmol of NNAL/mg of protein/min)
(Fig. 2a). Total bioactivation
(sum of the four -hydroxylation endpoint metabolites) ranged from 2.10
x 10-3 to 5.69 x 10-3%
-hydroxylation/mg of protein/min, representing 0.020 to 0.057 pmol
-hydroxylation/mg/min, which was less than 1% of the initial amount of
NNK over the 15-min incubation period (Fig.
2b). Total N-oxidation (sum of NNAL-N-oxide and
NNK-N-oxide) occurred at similar or slightly lower levels, with
N-oxidation being nondetectable in microsomes from patient 5JM
(Fig. 2c). Keto acid and keto
alcohol were detected in all of the patients' microsomal incubations, whereas
five of seven formed detectable levels of hydroxy acid, and six of seven
formed detectable levels of diol, NNAL-N-oxide, and
NNK-N-oxide (data not shown). Frequency distribution histograms
revealed the existence of high (6HM, 8HM, and 8JM) and low (3JM, 5JM, 7JM, and
9JM) NNK bioactivators, with significantly different mean total
-hydroxylation activities (i.e., 5.49 x 10-3 ±
0.17 x 10-3 and 2.45 x 10-3 ± 0.13
x 10-3 % -hydroxylation/mg/min, respectively,
P < 0.05, Student's t test)
(Fig. 2b). A similar trend for
the same groups of patients was observed for total N-oxidation, but
the difference fell just short of statistical significance (P =
0.054) (Fig. 2c). Individuals
forming the highest amounts of NNAL did not consistently form the highest
amounts of other metabolites (Fig.
2).
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When results from lung microsomes from different patients were grouped,
formation of keto acid was higher than that of all other -hydroxylation
endpoint metabolites, and formation of keto alcohol was significantly higher
than that of hydroxy acid, but not of diol
(Fig. 3). This may have been
due to higher levels of precursor for keto acid and keto alcohol (i.e., NNK)
than of hydroxy acid and diol (i.e., NNAL).
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There were no differences in total bioactivation or total N-oxidation in microsomes from current smokers (n = 4) versus former smokers (n = 3) (Student's t test, P > 0.05). Similarly, no differences were observed in formation of NNK metabolites between microsomes from male (n = 4) and female (n = 3) patients (P > 0.05, Student's t test).
Effects of Inhibitors on [5-3H]NNK Biotransformation: Grouped
Results. Most treatments had minimal effects on NNAL formation. However,
the absence of an NADPH-generating system virtually abolished NNAL production,
whereas incubations in the presence of 5.0 mM ABT formed significantly higher
amounts of NNAL relative to control (Fig.
4a). The absence of an NADPH-generating system or the presence of
5.0 mM ABT greatly decreased total -hydroxylation as well as total
N-oxidation (Fig. 4, b and
c). This trend held for individual NNK metabolites with the
exception of hydroxy acid and keto alcohol, where results where inconsistent
(data not shown). There were no other statistically significant differences in
incubations in the presence of either antibody or chemical inhibitors when
results were grouped. However, a trend toward inhibition of NNAL formation in
incubation mixtures containing SKF-525A was noted
(Fig. 4a).
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Effects of Inhibitors on [5-3H]NNK Bioactivation: Individual
Results. Substantial inhibition of total NNK bioactivation (sum of four
-hydroxylation endpoint metabolites) was observed in microsomes from
some patients. However, in a number of cases, the presence of either chemical
inhibitors or inhibitory antibodies actually increased total
-hydroxylation (Table
2). Also, effects of P450 enzyme-selective chemical inhibitors did
not always correlate with effects of specific P450 antibodies
(Table 2).
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Nonselective P450 inhibitors. CO lowered total
[5-3H]NNK -hydroxylation in four of five patients'
microsomes, by 6.3 to 53% (Table
2). SKF-525A decreased total -hydroxylation in lung
microsomes from three of five patients (6HM, 8HM, and 8JM, all of whom were
high bioactivators), with inhibition ranging from 38 to 47%
(Table 2). Additionally, at 1.0
mM, ABT, another nonselective P450 inhibitor, decreased total
-hydroxylation in all of five patients' microsomes by 21 to 56%, with
the greatest inhibition occurring in tissues from high bioactivators
(Table 2). Substantial
inhibition was observed with 5.0 mM ABT in microsomes from all patients.
Selective P450 inhibitors. Use of selective P450 enzyme inhibitors
revealed different patterns of enzyme contributions to NNK bioactivation for
different individuals. For patients 6HM and 8JM, both high bioactivators,
results suggested considerable CYP2A6 contribution to total
-hydroxylation (Table
2). CYP3A4 or CYP3A5 may have played a role in NNK bioactivation
in patient 6HM since inhibitory CYP3A4/3A5 antibody and the CYP3A inhibitor
TAO also decreased total -hydroxylation. For patient 8HM, another high
bioactivator, results suggested contributions particularly of CYP2B6, but also
of CYP2A6 and CYP2E1, to NNK total bioactivation
(Table 2). For most of the low
bioactivators, no clear-cut patterns with either selective inhibitory
antibodies or chemical inhibitors were observed, except that in microsomes
from patient 9JM, CYP2A6 appeared to play an important role
(Table 2).
When metabolism of NNK to specific metabolites was examined, levels of inhibition varied between patients and metabolites, and tended to be complementary when both immunoinhibition and chemical inhibition were compared in microsomes from the same individual (data not shown). However, effects of inhibitors did not consistently reveal that formation of any specific metabolite could be attributed to a specific P450 enzyme.
Non-P450 inhibitors. Notable inhibition of total NNK
-hydroxylation (i.e., >20%) by NDGA was observed in three patients'
microsomes (8HM, 7JM, and 9JM), with the greatest inhibition occurring in
microsomes from patient 7JM, in which inhibitory treatments did not reveal
contributions by specific P450 enzymes
(Table 2). Only one patient's
microsomes (9JM) demonstrated appreciable inhibition of total
-hydroxylation by INDO (i.e., 20%).
Effects of Inhibitors on Total N-Oxidation of [5-3H]NNK: Individual Results. Total microsomal N-oxidation (sum of NNK-N-oxide and NNAL-N-oxide) showed considerable variability between individual patients (Fig. 2c; Table 3).
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Nonselective P450 inhibitors. In three of five patients' microsomes, CO decreased total N-oxidation by 13 to 96%, and the nonselective P450 inhibitors SKF-525A and ABT (1.0 mM) inhibited total N-oxidation in four of five patients' microsomes by 36 to 88% and 7.3 to 73%, respectively (Table 3). Substantial inhibition was observed with 5.0 mM ABT in all microsomes containing detectable N-oxidation products in control incubates.
Selective P450 inhibitors. Although total N-oxide
production was highly variable, both enzyme-selective antibody inhibition and
chemical inhibition suggested patterns of enzyme-specific contributions. In
patient 8HM, the individual whose microsomes formed the highest amounts of
N-oxide metabolites, CYP2A6, CYP2B6, and CYP2E1 appeared to be
important contributors (Table
3). For patient 3JM, a relatively low producer of
N-oxides, both CYP2A6 and CYP2B6 were implicated
(Table 3). For patients 8JM and
9JM, CYP2A6 appeared to make a significant contribution. For patients 6HM,
7JM, and 8JM, antibodies and chemical inhibitors did not point to clear-cut
roles for specific P450 enzymes (Table
3). As in the case of -hydroxylation, in a number of
incubations N-oxidation activities in the presence of either chemical
inhibitors or inhibitory antibodies were unexpectedly increased compared with
control. Most notable was patient 5JM, an individual in whose microsomes
N-oxidation was nondetectable in control incubates
(Table 3). Additionally,
effects of enzyme-selective chemical inhibitors of total N-oxide
production did not always agree or correlate with effects of antibodies for
the same enzyme (i.e., CYP2B6).
Non-P450 inhibitors. Total N-oxide formation was inhibited by 15% or greater in microsomal incubations from five of seven patients' microsomes in the presence of NDGA, and four of seven in the presence of INDO (Table 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), characterization of its biotransformation in human lung
remains incomplete. In earlier studies, conclusions regarding the contribution
of various biotransformation enzymes to NNK metabolism were based on results
from only four individuals in total (Smith et al.,
1992,
1995). The principal aim of
the present study was to provide a more thorough examination of the
contributions of various biotransformation enzymes, particularly P450s, to NNK
metabolism in human lung.
Consistent with previous reports
(Castonguay et al., 1983; Smith
et al., 1992,
1995) and our findings in
isolated intact lung cells (Smith et al.,
1999), carbonyl reduction of NNK to NNAL was the major pathway for
NNK metabolism in microsomes from all seven individuals, and total
bioactivation (sum of four -hydroxylation endpoint metabolites)
represented less than 1% of the initial amount of NNK in the system. Despite
the fact that formation of both -hydroxylation and N-oxidation
metabolites occurred at very low rates, the enzymatic nature of metabolite
production was confirmed by heat lability, NADPH dependence, and inhibition of
their production, particularly by ABT.
For most patients, the overall balance between -hydroxylation
(bioactivation) and N-oxidation (detoxication) favored bioactivation.
Patterns of microsomal metabolite formation pointed out differences in
activity of different NNK biotransforming enzymes between patients. Keto acid
was the major -carbon hydroxylation metabolite in human lung
microsomes, reflecting potential formation of methylating species in smokers'
lungs.
NADPH dependence, coupled with pronounced and consistent inhibition of NNK
-hydroxylation and N-oxidation by ABT, suggests P450
contributions to NNK metabolism in all patients. In high bioactivators,
effects of CO and other inhibitors also support the importance of P450s to NNK
bioactivation and detoxication. The inconsistency with which CO and SKF-525A
caused inhibition relative to ABT is probably related to the particularly high
efficacy of ABT for P450 inhibition in lung microsomes. Indeed, Mathews and
Bend (1986) demonstrated
complete inhibition of P450 activity in rabbit pulmonary microsomes incubated
with 10 mM ABT. In the low human bioactivators, levels of P450 enzymes may
have been too low to observe consistent effects with other inhibitors.
Alternatively, Kim et al.
(1997) reported inhibition of
horseradish peroxidase-catalyzed benzo[a]pyrene bioactivation by ABT. Thus, it
is conceivable that ABT inhibits non-P450 enzymes that also contribute to
human pulmonary NNK biotransformation.
Our results indicate contributions of multiple P450 enzymes to NNK
metabolism in human lung microsomes, and these contributions vary between
individuals. Results from incubations in the presence of 8-MS or CYP2A6
inhibitory antibody suggest that CYP2A6 is involved in NNK bioactivation in
pulmonary microsomes from a number of people, including high and low
bioactivators. However, the recently cloned human P450, CYP2A13, cannot be
ruled out as a contributor to this activity, since CYP2A6 and CYP2A13 are
closely related and antibodies to CYP2A6 recognize both CYP2A6 and CYP2A13
(Su et al., 2000). CYP2A13
mRNA is expressed in human lung; the corresponding protein has catalytic
activity toward a number of CYP2A6 substrates and is more active than CYP2A6
in NNK bioactivation. The apparent importance of CYP2A6/13 is consistent with
the findings of Smith et al.
(1995), who suggested that NNK
bioactivation was catalyzed by CYP2A6 or an immunochemically related P450 in
human lung microsomes from three individuals.
Substantial involvement of CYP2B6 in NNK bioactivation in one individual's
pulmonary microsomes (patient 8HM) was consistent with the ability of
recombinant CYP2B6 to activate NNK to mutagenic species
(Smith et al., 1992;
Code et al., 1997), and with
immunoreactivity for CYP2B6 being detectable at varying levels in human lung
(Mace et al., 1998;
Gervot et al., 1999).
Results with an inhibitory CYP2E1 antibody suggested a role for CYP2E1 in
pulmonary NNK bioactivation in some patients' microsomes. Smith et al.
(1992) suggested no
involvement of CYP2E1 in NNK metabolism in human lung. However, that
conclusion was apparently based on results from only one subject, and
CYP2E1-catalyzed biotransformation of another nitrosamine,
N-nitrosodimethylamine, has been demonstrated at variable levels in
human lung microsomes (Botto et al.,
1994). Chemical inhibition studies to corroborate our
immunoinhibition results were not performed due to lack of availability of an
adequately selective chemical inhibitor. Thus, CYP2E1 contributions to human
pulmonary NNK bioactivation warrant further investigation.
Results from incubations with TAO and inhibitory antibodies to CYP3A4/3A5
suggested a minor role for CYP3A4 and/or CYP3A5 in NNK bioactivation in human
lung microsomes from only one individual (6HM). CYP3A5 was probably
responsible for NNK bioactivation, since it is the major CYP3A enzyme
expressed in the lung, with only 20% of individuals expressing CYP3A4
(Anttila et al., 1997). Also,
CYP3A5-mediated NNK -hydroxylation has been demonstrated
(Smith et al., 1992).
The limited effects of INDO on NNK bioactivation may have been due to
either inhibition of microsomal PHS-catalyzed -hydroxylation or,
possibly, to inhibition of P450s involved in NNK bioactivation. INDO is
metabolized primarily by CYP2C9 in human liver microsomes, with minor
contributions of CYP2C19, CYP1A2, and CYP2D6
(Nakajima et al., 1998). To
our knowledge, CYP2C8 is the only CYP2C class enzyme expressed in human lung
(Nakajima et al., 1994). Thus,
inhibition of CYP2C8 by INDO is plausible in human lung microsomes. CYP2C8 has
been implicated in the N-oxidation of NNK
(Smith et al., 1992), which
may account for the more pronounced inhibition of total N-oxidation
versus total bioactivation in incubations in the presence of INDO.
In the presence of an NADPH-generating system, NDGA inhibited NNK total
bioactivation and total N-oxidation in a number of patients'
microsomes. Interestingly, the greatest inhibition of bioactivation occurred
in microsomes from an individual with apparently limited P450 involvement
(Patient 7JM). Although NDGA has antioxidant properties and may inhibit some
P450s (Agarwal et al., 1991),
the concentration employed was selected to minimize these effects. However,
since LOXs are cytosolic and we have found that human lung LOX cannot
bioactivate NNK (Bedard et al.,
2002), the observed inhibition was likely occurring as a result of
actions on P450s or other microsomal peroxidases.
Our observation of apparent CYP2A6/13 catalysis of NNK N-oxidation
was unexpected, since NNK N-oxidation by recombinant CYP2A6 and
CYP2A13 was not observed (Smith et al.,
1992; Su et al.,
2000). CYP2B6 also appeared to contribute to N-oxidation,
whereas involvement of CYP3A4 and/or CYP3A5 in NNK N-oxidation in
human lung microsomes remains questionable, since results from CYP3A4/5
inhibitory antibody and TAO did not correlate.
In some microsomal incubations containing either chemical inhibitors or
inhibitory antibodies, we observed increased levels of NNK bioactivation and
N-oxidation. Large increases in a certain pathway could suggest
inhibitor-mediated stimulation of enzyme systems involved in NNK pulmonary
biotransformation, and some P450-selective chemical inhibitors activate
enzymes other than the P450 target for inhibition
(Ueng et al., 1997;
Ludwig et al., 1999). Although
paradoxical stimulation of biotransformation can occur with SKF-525A in intact
lung cells (Donnelly et al.,
1996; Smith et al.,
1999), to our knowledge, this phenomenon has not been reported for
the other inhibitors used in this study; also, similar effects are not likely
to be observed for inhibitory antibodies. Diversion of substrate from a
metabolic pathway that is sensitive to inhibition to one that is not is an
unlikely explanation, since the amount of NNK present was far in excess of the
amount that would be made available by inhibition of a competing metabolic
pathway. Nonuniform constitution of human lung microsomes between incubates
may contribute to apparent stimulation by inhibitors. Human lung microsomes
can be contaminated by particulate matter such as carbon or tar, accumulated
from years of cigarette smoking, which might affect how NNK biotransformation
activities are expressed in different incubates. The fact that apparent
stimulation by inhibitors occurred almost exclusively in microsomes with low
control activities supports this explanation.
The consistent increase in NNAL formation in the presence of 5.0 mM ABT,
which averaged 18%, could not be accounted for by increased NNK
availability due to inhibition by NNK of -hydroxylation and
N-oxidation, and suggests that ABT stimulates microsomal
11ß-hydroxysteroid dehydrogenase (11ß-HSD), the carbonyl reductase
responsible for the majority of NNAL formation in human lung
(Maser et al., 2000). Although
not statistically significant (P = 0.102), the trend toward decreased
NNAL formation in human lung microsomes with 1.0 mM SKF-525A
(Fig. 4a) was consistent with
sensitivity of 11ß-HSD and/or other carbonyl reductases to SKF-525A
(Smith et al., 1999).
Interindividual variability in NNK biotransformation could potentially be attributed to factors including therapeutic intervention prior to surgery and occupational exposure to biotransformation enzyme inducers and inhibitors. However, no associations between drug treatments and microsomal NNK biotransformation were apparent. We also saw no evidence of smoking status or sex-related differences in microsomal NNK metabolism.
CYP2A6 and CYP2A13 are polymorphic, with high and low activity-variant
alleles. Despite the apparent importance of CYP2A6/13 in NNK metabolism in
human lung microsomes, the interindividual differences observed in NNK
metabolism are unlikely to be attributable to the presence of identified
mutant CYP2A6 alleles or CYP2A13 alleles because the combined frequency of
alleles in white populations conferring poor metabolizer phenotypes is less
than 3.5% and 2.0%, respectively (Raunio
et al., 2001; Zhang et al.,
2002). Nonetheless, the observation of high and low NNK
bioactivators is consistent with the proposal of Castonguay et al.
(1983) of the existence of a
subset of individuals with high NNK bioactivation and, hence, elevated
susceptibility to NNK-induced carcinogenesis. Elucidation of the biological
processes differentiating these individuals will require further study.
In conclusion, results from the present study indicate that several P450 isozymes contribute to NNK biotransformation in microsomes prepared from human peripheral lung. It is likely that the relative contributions of these enzymes vary between individuals. CYP2A6 and/or CYP2A13 appear to play a significant role in NNK oxidation in lungs of many humans. Other P450 isozymes including CYP2B6, CYP2E1, and CYP3A5 appear to play a role as well, but not in all humans.
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Acknowledgments |
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Footnotes |
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1 Abbreviations used are: NNK,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; P450, cytochrome P450; LOX,
lipoxygenase; PHS, prostaglandin H synthase; SKF-525A, proadifen
hydrochloride; ABT, 1-aminobenzotriazole; INDO, indomethacin; 8-MS,
8-methoxypsoralen; NDGA, nordihydroguaiaretic acid; ORP, orphenadrine
hydrochloride; TAO, troleandomycin; MB,
N--methylbenzyl-1-aminobenzotriazole; BBT,
N-benzyl-1-aminobenzotriazole; ANOVA, analysis of variance; CO,
carbon monoxide; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase. 
Address correspondence to: Dr. Thomas E. Massey, Department of Pharmacology and Toxicology, Queen's University Kingston, ON K7L 3N6 Canada. E-mail: masseyt{at}post.queensu.ca
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,
, and 
); metabolite VII is the O-glucuronide of NNAL.
Oxobutyl diazohydroxides (
