Eppley Institute for Research in Cancer (S.C.C., L.Z., S.S.M.) and
Departments of Biochemistry and Molecular Biology and of Pharmaceutical
Sciences (S.S.M.), University of Nebraska Medical Center, Omaha,
Nebraska; and Wadsworth Center, New York State Department of Health and
School of Public Health (X.D.), State University of New York, Albany,
New York
 |
Introduction |
There
are especially high rates of squamous cell carcinoma of the esophagus
in South Africa, Iran, and China, and the incidence of esophageal
adenocarcinomas is increasing rapidly in Western countries (Mirvish,
1995
). Nitrosamines are probably initiating agents for esophageal
cancer, especially squamous cell carcinoma, for several reasons (Magee,
1989; Mirvish, 1995). Esophageal cancer is induced in rats by
nitrosamines, such as methyl-n-pentylnitrosamine (MPN2) (Fig. 1),
methylbenzylnitrosamine (MBZN), N-nitrosopiperidine, and the
tobacco-specific compound N'-nitrosonornicotine (NNN), but
not by dimethylnitrosamine (DMN) (Preussmann and Stewart, 1984). NNN
and related nitrosamines that occur in tobacco smoke are the probable
initiators of esophageal cancer in smokers (Hecht and Hoffmann, 1989;
Magee, 1989). Asymmetric dialkylnitrosamines similar to MPN that are
derived from moldy corn could contribute to the etiology of esophageal
cancer in China and South Africa (Mirvish, 1995). The corn mold
Fusarium moniliforme was reported to convert
iso-pentylamine to
N-methyl-N-iso-pentylamine, which is
readily nitrosated to yield iso-MPN; however, we failed to confirm this result (Mirvish, 1995). Esophageal exposure to
nitrosamines could occur by direct absorption while foods are being
swallowed (Haorah et al., 1999). Subjects from Chinese counties with
high incidences of esophageal cancer showed elevated urinary excretions of N-nitrosoproline, an indicator of in vivo nitrosation (Wu
et al., 1993). A case-control study in Linxian found significant associations of the c1/c1 genotype of the cytochrome P450 isozyme CYP2E1 with esophageal hyperplasia and cancer (Tan et al., 2000). Possibly, the c1/c1 phenotype of CYP2E1 metabolizes nitrosamines less
efficiently in the liver than does the c2/c2 phenotype, hence allowing
more nitrosamine to reach the esophagus.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Metabolism of MPN to yield formaldehyde and
a pentylating agent (route a), PENT and a methylating agent (route b),
and stable hydroxy-MPNs (route c).
|
|
Nitrosamines are activated by hydroxylation on the carbon atom(s)
adjacent to the nitrosamine group. In the rat esophagus, this reactions
is catalyzed by one or more unidentified cytochrome P450s (Murphy and
Spina, 1994; Chen et al., 1999a; Gopalakrishnan et al., 1999). MPN
metabolism by depentylation to give pentaldehyde (PENT) and an agent
that methylates DNA bases (route b of Fig. 1) is a more likely
activation mechanism than route a of Fig. 1, in which MPN is
demethylated to give formaldehyde and a pentylating agent, because 7- and O6-methylguanine, but not 7- and
O6-pentylguanine, were detected in
esophageal DNA of rats treated with MPN (Huang et al., 1993). Rat liver
microsomes produced formaldehyde, PENT, 4-hydroxy-MPN, and nitrite from
6 mM MPN (Ji et al., 1989). Human esophageal and liver microsomes
metabolized 6 mM MPN to yield formaldehyde and PENT (Huang et al.,
1992). When [3H]PENT production from
[pentyl-3H]MPN was assayed; the
Km values for MPN depentylation by rat esophageal and liver microsomes, rat CYP2E1, human CYP2E1, and human
CYP2A6 [a liver, nasal, and lung P450 (Su et al., 2000)] were 64, 610, 210, 115, and 17 µM, respectively (Chen et al., 1999a). MPN
metabolism by rat and human esophageal microsomes was strongly
inhibited by CO, a P450 inhibitor (Huang et al., 1992; Chen et al.,
1999a).
In the present study, we performed further experiments on depentylation
by human and rat microsomes of (in standard runs) 100 µM
3H-labeled MPN of high specific activity (Chen et
al., 1999a). Even if human esophagus actively depentylated MPN, this
would indicate that nitrosamines might preferentially induce human
esophageal cancer only if this activity was high relative to that for
other organs. Therefore, we compared the depentylating activity of
human esophageal microsomes with that of microsomes prepared from other human organs. For similar reasons, we also determined the MPN depentylation activity of microsomes from six rat organs, in addition to the previously studied (Chen et al., 1999a) esophagus and liver.
The unusually low Km of 17 µM for MPN
depentylation by CYP2A6 (Chen et al., 1999a) suggested that a CYP2A
enzyme was responsible for MPN metabolism by rat esophagus. CYP2A3, a
rat ortholog of human CYP2A6, occurs mainly in nasal mucosa and, to
minor extents, in the lung and esophagus (Su et al., 1996;
Gopalakrishnan et al., 1999) and catalyzed MBZN activation with an
apparent Km of only 0.6 µM (Von Weymarn
et al., 1999). Hence, CYP2A3 might also activate MPN in the rat
esophagus and nasal cavity where MPN induces tumors (Bulay and Mirvish,
1979). Accordingly, we also investigated MPN depentylation by CYP2A3
(Liu et al., 1996). Finally, we examined the effect of adding P450
reductase on MPN metabolism by rat esophageal microsomes.
| |
Materials and Methods |
Synthesis of [3H]MPN.
1-Bromo-2-pentene or 5-bromo-1-pentene was reacted with methylamine to
give methyl-2-pentenylamine or methyl-4-pentenylamine, respectively
(Chen et al., 1999a). Hydrogenation with tritium at Moravek
Biochemicals (Brea, CA) yielded
[2,3-pentyl-3H]- or
[4,5-pentyl-3H]methylpentylamine. As required,
about 100 mCi of the amine was nitrosated to give
[2,3-pentyl-3H]MPN or
[4,5-pentyl-3H]MPN (Chen et al., 1999a). The
[3H]MPN was purified by thin layer
chromatography on alumina developed with hexane-ether-acetic acid
(40:60:5) and, on the day of the experiment, by reaction with
semicarbazide to remove PENT, followed by thin layer chromatography on
alumina developed with hexane-ether (4:6) (Chen et al., 1999a). For
final MPN concentrations of <50 µM, the contribution of
[3H]MPN to the total MPN was significant and
was determined by gas chromatography-thermal energy analysis of the
[3H]MPN. Specific activity of the
[3H]MPN was typically 160 µCi/µmol.
Tissues Studied.
See Acknowledgments for the suppliers of the human tissue
specimens. Each specimen weighed 5 to 50 g. All tissues appeared normal and did not include obvious tumors. The position of esophageal specimens within the esophagus was not reported. Most tissues were
obtained from autopsies, all of which were performed 
6 h after death. At autopsy, the tissues were snap-frozen in liquid N2 and shipped in dry ice. Other tissues were
obtained during operations and handled similarly. Tables
1 and 2 summarize the pathology reports. Rat esophagi of freshly killed adult male
Sprague-Dawley rats were obtained from Harlan Bioproducts for Science
(Indianapolis, IN), who stripped away the outer connective tissue,
flash-froze the tissues in liquid N2, and mailed
them in dry ice (Chen et al., 1999a). Other rat tissues were obtained
from male Sprague-Dawley rats 6 to 8 weeks old (Sasco, Omaha, NE) that
were euthanized with CO2. Tissues were stored at
70°C, most for <6 months, before use.
View this table:
[in this window]
[in a new window]
|
TABLE 1
Depentylation of 100 µM MPN by microsomes from human tissues
Causes of death or reasons for operation were: for esophagus, see Table
2; for stomach samples, two stomach cancer, one brain hemorrhage, and
one cardiac arrest; for small intestine samples, one pancreatic cancer,
prostate cancer, one obstruction of small intestine, and one unknown
(last two from operations); for colon samples, two colon cancer (one
removed at operation), one rectal adenoma, and one homicide; for liver
samples, one multiple injuries, one arteriosclerosis, two heart
disease, and one hemangioma; for kidney samples, one heart disease, one
bladder cancer, and three renal cancers; and for lung samples, one
myocardial infarction, one lung vein aneurysm, and two lung cancers.
All skin samples were obtained from operations to remove excess
abdominal fat.
|
|
Isolation of Microsomes.
From human tissues
Each microsome sample was obtained from a different donor. All
operations were performed in a cold room at 5°C. The stored tissues
were frozen in liquid N2 without prior thawing,
wrapped in cheese cloth, and pulverized with a hammer. Portions (5-10 g) of softer tissues (liver, kidney, and lung) were dispersed with a
Tissumizer (Tekmar-Dohrmann, Mason, OH) in 0.1 M potassium phosphate buffer (pH 7.4, 3 ml/g of tissue) containing 0.1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride (buffer A) and
further homogenized with a motor-driven Potter-Elvejhem tissue
grinder (Labglass, Vineland, NJ) fitted with a Teflon piston (Chen et
al., 1999a). For harder tissues (esophagus, stomach, intestines, and
skin), 5 to 10 g pieces of frozen tissue were ground to a fine
powder with a mortar and pestle precooled with liquid
N2. The powder from human esophagus was
homogenized in a chilled 15-ml Tenbroeck Pyrex-glass tissue
homogenizer (Scientific Products, Edison, NJ) in 3 ml/g of tissue of
0.1 M sodium pyrophosphate buffer (pH 7.4) containing 1 mM EDTA, 1 mM
dithiothreitol, and 5 mM phenylmethylsulfonyl fluoride (buffer B)
(Murphy and Spina, 1994; Chen et al., 1999a). The homogenizing medium
for all other human tissues was buffer A (3 ml/g of tissue). Microsome
fractions were isolated by differential centrifugation (Huang et al.,
1992), resuspended in buffer A or B containing 20% glycerol, and
analyzed for protein by the Lowry method (Lowry et al. 1951). The
suspensions were stored at 70°C, most for <3 months, before use.
The activity of several samples that were re-examined was mostly
unaffected after storage for 2 years.
From rat tissues.
Microsomes from 16 to 18 rat esophagi at a time were prepared as before
(Murphy and Spina, 1994; Chen et al., 1999a). In brief, esophagi frozen
in liquid N2 were crushed in a Bessman
tissue pulverizer (Fisher Scientific, Springfield, NJ) and gently
homogenized in buffer B (1 ml/esophagus) using a Tenbroeck homogenizer.
Microsomes from other rat tissues were prepared as described for the
corresponding human tissues.
Metabolic Experiments.
These followed methods used before (Huang et al., 1992; Chen et al.,
1999a). Incubations were performed in 5-ml stoppered disposable
polystyrene round-bottom tubes with 12 to 16 tubes per experiment. In
standard runs, we mixed 100 mM potassium phosphate buffer (pH 8) with
10 mM MgCl2 and 5 mM semicarbazide HCl (the pH
was then 7.4). We then added 10 to 30 µl of a microsomal suspension containing 100 to 200 µg of protein, MPN (20 to 2000 µM, 2-10 µCi/tube) and, finally, an NADPH-generating system (2 mM NADP, 10 mM
glucose 6-phosphate, and 2 units of glucose-6-phosphate dehydrogenase;
final concentrations are indicated in all cases). The total volume was
0.5 ml, and the final pH was 7.4. The mixtures were incubated for 20 min at 37°C with shaking twice per second [reaction rates are
constant over this time (Chen et al., 1999a)]. Appropriate control
tubes were included. Duplicate tubes were used for each condition.
Reactions were stopped by adding Ba(OH)2 and
ZnSO4, and PENT 2,4-dinitrophenylhydrazone was
prepared and separated by high-pressure liquid chromatography on an
analytical C18 column developed with a gradient
rising from 50 to 70% ethanol in water (Chen et al., 1999a). The PENT
hydrazone showed a retention time of 21 min (Fig.
2).
We collected 1-ml fractions and counted them in a Beckman
scintillation counter (Beckman Coulter, Inc., Fullerton, CA) (Chen et
al., 1999a) or counted the eluate directly with a flow scintillation analyzer (500 TR Series, Packard, Meriden, CT) with integration of peak
areas by the Flo-one program with "force horizontal" baseline (Fig.
2). Peak width of the PENT hydrazone was about 3 min. Counts per minute
for blanks without microsomes were subtracted from experimental values.
The results, expressed as counts per minute, were used to calculate
PENT yield from MPN. The yield at 20 min was expressed as rate per
minute, assuming that the rate did not change for 20 min, as found in a
test with phenobarbital-induced rat liver microsomes (Chen et al.,
1999a). Yields were graphed with a Prism program (GraphPad Software,
San Diego, CA) for determining nonlinear regression curves.
Km and Vmax
were obtained from the best-fitting straight lines for Lineweaver-Burke
plots using Excel 4.0 (Microsoft, Redmond, WA).
| |
Results |
Comments on Methods.
Most of these studies were carried out using
[2,3-pentyl-3H]MPN, but some of the earlier
tests used [4,5-pentyl-3H]MPN. As explained
previously (Chen et al., 1999a), the yield of
[3H]PENT should not depend on which isomer is
employed because MPN activation does not involve compounds with labile
hydrogen at C-2 to C-5 and because the product PENT was not subjected
to alkaline conditions and hence should not have enolized. The two
isomers seemed to give similar results, but the
3H 2,3-pentyl isomer of MPN seemed to be more
readily synthesized and more stable than the 3H
4,5-pentyl isomer. The efficiency of the conversion of PENT to its
hydrazone is 23% (Chen et al., 1999a). As before (Chen et al., 1999a),
the results were not corrected for this loss.
MPN Metabolism by Human Microsomes.
Table 1 shows the depentylation rates when human microsomes were
incubated with 100 µM MPN. This MPN concentration was used in
standard runs even though it is lower than some of the
Km values for human esophagus and liver
(Table 2) and hence did not give accurate comparisons of the maximum
rates. A standard MPN concentration of 100 µM was used for three
reasons. 1) The use of low MPN levels brought the MPN level closer to
those of nitrosamines that people are exposed to. 2) In studies on
microsomes, if we had raised the standard MPN level to 300 µM, for
example, we would probably have recruited P450 isozymes that metabolize
MPN with relatively high Km values. In
fact, this probably occurred in our previous study with 6 mM unlabeled
MPN (Huang et al., 1992). 3) If we had raised the standard MPN
concentration, we would have obtained a reduced percent yield of PENT
because absolute PENT yield would not have been much higher than that
obtained with 100 µM. However, we would have had to use
[3H]MPN of a higher specific activity to obtain
reasonable counts for the PENT hydrazone peak, and this would have
raised the background counts. In fact, this was somewhat of a problem
when high-MPN concentrations were used in the measurements of
Km.
Mean depentylation activity for human tissues was highest for the
liver, moderate for kidney, small intestine, stomach, and colon, low
for esophagus (24% of that for the liver), and especially low for lung
and skin. Esophageal microsomes from 14 subjects were tested (Table
3). The results for different samples of
the same batch of microsomes showed reasonable agreement with most standard errors <30% of the mean values. In contrast, the mean activity for esophageal microsomes from different subjects varied from
0 to 4.1 pmol of PENT/mg of protein/min. Of the 14 esophagi examined,
six were apparently normal tissue from individuals with cancer. The
only noted relationship to the cause of death was that none of the
cancer cases showed esophageal activity 
1.8 pmol of PENT/mg/min.
Table 2 shows the kinetic rate constants (apparent
Km and Vmax)
and metabolic rates for the depentylation of
[3H]MPN by human microsomes from three
esophagi, two livers, and one kidney. These results were obtained using
20 to 2000 µM MPN and are compared in Table 2 with our published
values (Chen et al., 1999a) for rat esophagus and liver.
Lineweaver-Burk plots were constructed (e.g., see Fig.
3). The Km
values for microsomes from human esophagus and liver were 80 to 160 µM and were 1.25 to 2.5 times the Km for
rat esophageal microsomes. The Vmax for human esophageal microsomes was 17 to 75% of that for rat esophageal microsomes. Coumarin (0.4 mM) inhibited the depentylation of 100 µM
MPN by 37 to 62% in tests on microsomes from human esophagus, liver,
kidney, and colon (Table 4). As before
(Chen et al., 1999a), coumarin was incubated with the microsomes for 15 min before the addition of MPN. The effect of coumarin on human lung
microsomes could not be examined because of their low activity (Table
1).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Kinetics of [3H]MPN
depentylation by microsomes (100 µg of protein/tube) from human
esophagus no. 1 (Table 3).
This figure shows a Lineweaver-Burk plot of 1/S versus
1/V, where S = substrate
concentration and V = rate of reaction. Each point
gives the results for an individual tube. This figure indicates a
Km of 80 µM and a
Vmax of 15 pmol/mg of protein/min.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 4
Effect of 0.4 mM coumarin on the depentylation of 100 µM MPN by human
and rat microsomes
Phosphate buffer, MgCl2, semicarbazide, and water (total volume
about 300 µl, see Materials and Methods), microsomes
(10-20 µl), and 40 µl of 5 mM coumarin in water were mixed and
incubated for 15 min at room temperature with occasional stirring.
Control mixtures were incubated similarly but without addition of
coumarin. [3H]MPN, unlabeled MPN, and NADPH-generating system
were added to give a final volume of 500 µl and a MPN level of 100 µM. Incubation was then performed, and depentylation was assayed as
described under Materials and Methods.
|
|
MPN Metabolism by Rat Microsomes and CYP2A3.
Table 5 shows depentylation rates when
100 µM MPN was incubated with microsomes from eight rat tissues. The
average depentylation rate of 14.5 pmol/mg of protein/min for esophagus
was exceeded only by that for liver, the order of activity being
liver > esophagus > lung > colon > forestomach > glandular stomach > kidney > small intestine. Coumarin inhibited the depentylation of 100 µM MPN by 55%
when rat esophageal microsomes were tested but had no effect with rat
liver and lung microsomes (Table 4).
View this table:
[in this window]
[in a new window]
|
TABLE 5
Rate of depentylation of 100 µM MPN by microsomes from rat tissues
Results were obtained with four preparations of esophageal microsomes
and one preparation each of microsomes from the other listed tissues.
Each microsomal preparation was isolated from the tissues of at least
two rats.
|
|
We examined MPN depentylation by rat CYP2A3 expressed in Sf-9 insect
cells using a baculovirus (Liu et al., 1996). The CYP2A3 was incubated
with a 4-fold molar excess of rabbit P450 reductase because this ratio
produced maximum testosterone metabolism by this P450 (Liu et al.,
1996). Under these conditions, the Km was 8 µM, and Vmax was 70 pmol/nmol of P450/min
(Fig. 4). Coumarin inhibition of MPN
depentylation by rat CYP2A3 was then investigated. The results were
expressed as Dixon and Cornish-Bowden plots, i.e., plots of
1/V, and S/V, respectively, against
inhibitor (coumarin) concentration, where S = substrate
concentration and V = rate of reaction (Fig.
5). The MPN concentration where the two
lines met in the Dixon plot indicated an apparent
Ki of 4 µM, and the nearly parallel lines
in the Cornish-Bowden plot indicated that the inhibition was
competitive (Cornish-Bowden, 1974).
Finally, we determined the effect of adding varied amounts of P450
reductase on the depentylation of 100 µM MPN by rat esophageal and
liver microsomes. Addition of the reductase increased PENT yield from
100 µM MPN by up to 90% (esophagus) and 54% (liver) (Fig.
6). In the case of esophagus, 80 pmol of
reductase increased depentylation by only 15%, whereas 160 pmol of
reductase increased the yield by 74%. Incubation of MPN with 300 pmol
of reductase in the absence of P450 did not produce a significant yield
of PENT.
| |
Discussion |
MPN Metabolism in Human Tissues.
Although human esophageal microsomes generally exhibited low activity
for MPN dealkylation, microsomes from 4 of the 14 esophagi showed
relatively high activities of 3.3 to 4.1 pmol of PENT/mg of protein/min
for the metabolism of 100 µM MPN (Table 3), about half the mean value
of 6.6 pmol/mg/min for human liver (Table 1). If this high activity
occurred only in mucosal microsomes [likely because nitrosamines are
chiefly activated in rat esophagus by basal mucosal cells (Koenigsmann
et al., 1988)], the activity of the mucosal microsomes may have been
much higher than the values reported here because the mucosa is only 15 to 20% of the total human esophagus, all of which was used to prepare
the microsomes. The Km and
Vmax values for human esophageal microsomes
were similar to those for human liver microsomes and had somewhat
higher Km values for human than for rat
esophageal microsomes (Table 2). The relative activity of two enzyme
preparations at low-substrate concentrations is indicated by the
relative
Vmax/Km ratios,
which should equal the relative activities at a single, low-substrate concentration. In agreement with this concept,
Vmax/Km for
esophagus no. 1 divided by the same ratio for esophagus no. 10 was 6.8 (Table 2), and the depentylation rate for 100 µM MPN in esophagus no. 1 divided by that for esophagus no. 10 (Table 3) showed a fairly similar value of 9.1. Other comparisons of
Vmax/Km for the
different microsome samples also showed reasonable agreements with
comparisons of the rates for 100 µM MPN. The unusually high
Vmax of 74 pmol/mg/min for human kidney
(Km, 1330 µM) might have been due to the
high-MPN level used with this tissue and, hence, the recruitment of
additional P450s not involved at low-MPN concentrations.
With regard to the P450s known to occur in human esophagus, evidence
was found for CYP1A1/1A2 in all tested human esophagi and for CYP2B6 in
12 of 25 esophagi (Murray et al., 1994; Nimura et al., 1997). In human
esophagus, Lechevrel et al. (1999) detected the mRNAs of P450s 1A1,
1A2, 2A, 2E1, 3A5, 4A1, and 4B1 and expression of P450s 1A1/1A2, 2A6,
2E1, 3A4/5, and 4A. Barrett's esophagus showed expression of P450s
1A2, 2E1, 2C9/10, and 3A4 (Hughes et al., 1999). All of 22 samples of
human esophageal microsomes metabolized NNN and DMN (Smith et al.,
1998). NNN activation was reduced 20 to 30% by a CYP3A inhibitor.
Coumarin 7-hydroxylase activity (characteristic of some CYP2A enzymes)
was not detected. It was concluded that the esophageal metabolism of
NNN and DMN was catalyzed by CYP3A4 and CYP2E1, respectively (Smith et
al., 1998). MBZN is metabolized in human liver chiefly by CYP2A6 and
CYP2E1 (Morse et al., 1999). DMN, diethylnitrosamine, and
methylphenylnitrosamine were dealkylated by CYP2A13, a human lung
enzyme (Su et al., 2000). Our finding that 0.4 mM coumarin inhibited
MPN metabolism by 37 to 62% in the human esophagus, liver, kidney, and
colon (Tables 3 and 4) indicates that these tissues may contain CYP2A enzymes.
Our results with human tissues, which were frozen <6 h after death,
could have been affected by postmortem changes (e.g., freezing human
liver samples, thawing them, and then keeping them at room temperature
for 5 h led to 90% losses of the P450s) (Yamazaki et al., 1997).
Here, frozen tissue samples were thawed at 6°C and immediately
homogenized and centrifuged to obtain the microsomes. Therefore, most
of the P450 activity in the frozen human tissues probably persisted in
the microsomes. Also, the microsomes were stored in 20% (2.2 M)
glycerol to increase their stability. Glycerol inhibits DMN metabolism
by rat CYP2E1 with a Ki of 53 mM (Yoo et
al., 1987) and, hence, might have inhibited MPN metabolism. However,
this effect is known to occur only with CYP2E1, which probably is not
involved in MPN metabolism by rat esophagus. No differences were found
when MPN depentylation by rat esophageal microsomes and
phenobarbital-induced liver microsomes, suspended in 20% glycerol
(concentration in incubation medium, 40-130 mM), was compared with MPN
metabolism in glycerol-free medium (Chen et al., 1999a).
Comparison with MPN Metabolism in Rat Tissues and by the Rat P450,
CYP2A3.
Rat esophageal microsomes showed apparent
Km values of 64, 3.8 to 5.1, and 49 µM
for the activation of MPN, MBZN, and NNN, respectively (Murphy et al.,
1990; Murphy and Spina, 1994; Chen et al., 1999a; Von Weymarn et al.,
1999). The activity of these microsomes for MPN metabolism was higher
than that of microsomes from six other rat organs and was exceeded only
by those from the liver (Table 5). This supports the view that these
nitrosamines induce tumors of the rat esophagus because they are
preferentially activated there. The main reason why these nitrosamines
do not induce liver cancer despite extensive metabolism in that organ may be the slow rate of cell turnover in the liver [basal cells of the
mouse esophageal mucosa have half-lives of 4.5 days (LeBlond et al.,
1964)]. The 10-fold higher Km for MPN
compared with that for MBZN is consistent with the approximately 10 times lower carcinogenicity of MPN for the rat esophagus (Haorah et
al., 1999). Nevertheless, MPN is nearly as specific an esophageal
carcinogen as MBZN (Preussmann and Stewart, 1984).
MPN metabolism by rat esophageal microsomes was inhibited up to 65% by
coumarin (Chen et al., 1999a). Most of this inhibition occurred only
when coumarin was preincubated with the microsomes for 15 to 30 min
before the incubation with MPN, which was also found for MBZN
metabolism by these microsomes (Chen et al., 1999a; Von Weymarn et al.,
1999). These results support the view (Von Weymarn et al., 1999) that
an active metabolite of coumarin causes the inhibition. Coumarin
inhibited the metabolism of other nitrosamines by rat esophageal and
nasal microsomes (the latter are rich in CYP2A3) (Bereziat et al.,
1995; Patten et al., 1998; Von Weymarn et al., 1999), the mouse liver
enzyme, CYP2A5 (Negishi et al., 1989), and the human enzyme CYP2A6,
where coumarin showed a Ki of 7.5 µM for
the inhibition of MPN depentylation (Chen et al., 1999a). The
hypothesis (Gopalakrishnan et al., 1999) that the rat esophageal P450
that activates esophagus-specific nitrosamines is a CYP2A isozyme is
supported by these observations and by findings that CYP2A3 metabolized
MPN and MBZN with Km values of only 8 and
0.63 µM, respectively (Fig. 4; Von Weymarn et al., 1999). However,
the P450 in rat esophageal microsomes is not CYP2A3 because these
microsomes contain only low levels of this P450 (Gopalakrishnan et al.,
1999) and, unlike CYP2A3, are poor catalysts of coumarin 7-hydroxylation (Chen et al., 1999a; Gopalakrishnan et al., 1999). Our
findings that CYP2A3 metabolism of MPN showed a
Km of 8 µM (Fig. 4) and was competitively
inhibited by coumarin with a Ki of 4 µM
(Fig. 5) extends the list of nitrosamines metabolized by CYP2A3 and the
list of CYP2A enzymes (which now includes P450s 2A3, 2A5 and 2A6)
that are inhibited by low levels of coumarin.
Our observation that added P450 reductase increased the rate of MPN
metabolism by up to 90% in the rat esophagus and 54% in the rat liver
(Fig. 6) suggests that the level of this enzyme limits nitrosamine
metabolism in these tissues. Similar findings were reported for
warfarin metabolism by rat liver microsomes (Kaminsky and Guengerich,
1985). The activities of P450s in microsomes are far lower than those
of the same levels of purified P450s incubated with excess reductase,
probably because different P450s in microsomes compete for a limited
amount of reductase (Kaminsky and Guengerich, 1985).
General Comments.
The highly sensitive assay of [3H]MPN
depentylation used here and previously (Chen et al., 1999a) offers a
useful method for studying the metabolism of certain CYP2A isozymes and
of CYP2E1 in small samples of microsomes or of the individual P450s.
Our results using 100 µM MPN resemble our preliminary results for four human esophagi using 6 mM MPN (Huang et al., 1992) in that the
mean rate of MPN metabolism for human esophagus in both studies was 9 to 11% of that for rat esophagus and was <25% of that for human
liver. Human esophagus did not generally show a high level of MPN
metabolism compared with other human organs. The relatively high MPN
metabolism in 4 of 14 human esophagi could be due to differences in
tissue handling or to intrinsically high-P450 activity, which could
increase the susceptibility to esophageal carcinogenesis by
unsymmetrical dialkylnitrosamines.
In human esophagus, CYP2E1 may be involved in the metabolism of
nitrosamines, especially DMN, because this P450 exhibited polymorphism
associated with esophageal cancer (Tan et al., 2000), and rat and human
CYP2E1 showed apparent Km values of 20 to
22 µM (Yoo et al., 1990; Patten et al., 1992) for DMN demethylation, and 210 and 115 µM, respectively, for MPN depentylation (Patten et
al., 1992; Chen et al., 1999a). Human esophageal microsomes activated
DMN (Smith et al., 1998), Therefore, if nitrosamines cause human
esophageal cancer in high-incidence areas of China and South Africa
where cigarette smoking is probably not a factor (Mirvish, 1995), then
symmetrical dialkylnitrosamines might be at least as important in the
etiology as unsymmetrical dialkylnitrosamines, such as MPN and MBZN. In
contrast, unsymmetrical dialkylnitrosamines are the most potent
carcinogens in the rat esophagus (Preussmann and Stewart, 1984).
However, the tobacco-specific cyclic nitrosamine NNN is metabolized by
both human and rat esophageal microsomes (Murphy and Spina, 1994; Smith
et al., 1998), is carcinogenic for the rat esophagus (Preussmann and
Stewart, 1984), and is probably involved in the etiology of esophageal
cancer in smokers (Hecht and Hoffmann, 1989). The finding that rat
esophagus showed high activity for MPN metabolism relative to that for
most other rat organs seems to explain why MPN and, perhaps, certain
other nitrosamines induce esophageal cancer in rats and is attributed
to an unidentified esophagus-specific P450. This might be a homolog of
CYP2A13, a human lung P450 that demethylated methylphenylnitrosamine, a
rat esophageal carcinogen (Su et al., 2000), or might be CYP2B21, a
novel P450 expressed in rat esophagus (Brookman-Anissah et al., 2001).
We thank Lawrence Schopfer (Eppley Institute) for advice about enzyme
kinetics, Peter Guengerich (Vanderbilt University, Nashville, TN) for
advice about the effects of P450 reductase, and the National Disease
Research Interchange (Philadelphia, PA) and the Western Division of the
Cooperative Human Tissue Network (Department of Pathology, Case Western
Reserve University, Cleveland, OH) for supplying the human tissue samples.
This research was supported by Grant RO1-CA-35628 and core
Grant P30-CA-36727 from the National Cancer Institute, Grant
RO1-ES-07462 from the National Institute for Environmental Health
Sciences, and Grant 97B-125 from the American Institute of Cancer Research.
S. S. Mirvish, Eppley
Institute for Research in Cancer, University of Nebraska Medical
Center, Omaha, NE 68198-6805. E-mail: smirvish{at}unmc.edu
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
-hydroxylates N'-nitrosonornicotine.
Carcinogenesis
15:
2709-2713
, tests with 0.53 µM MPN; 
, tests with 2.65 µM MPN.
, esophageal
microsomes;