[CANCER RESEARCH 44, 2885-2891,
July 1984]
Non-Alcohol Dehydrogenase-mediated Metabolism of Methylazoxymethanol
in the Deer Mouse, Peromyscus maniculatus1 2
Emerich S. Fiala,3 Nancy Caswell, Ock Soon Sohn, Michael R. Felder, G. David McCoy, and John H. Weisburger
Naylor Dana Institute for Disease Prevention, Valhalla, New York 10595 [E. S. F., N. C., 0. S. S., J. H. W.¡;Department of Biology, University of South Carolina,
Columbia, South Carolina 29208 [M. R. F.J; and Department of Epidemiology and Community Health, Center for the Environmental Health Sciences, Cleveland, Ohio 44106
[G. D. M.]
and MAMOAc,
ABSTRACT
The concept that alcohol dehydrogenase (ADH) is involved in
the metabolism of methylazoxymethanol (MAM) was examined
in a model consisting of two strains of the deer mouse, Pero
myscus maniculatus, one of which has a normal complement of
the enzyme [ADH(+)J, and the other, which completely lacks it
[ADH(-)]. Both the ADH(+) and the ADH(-) strains rapidly
metabolized [14C]MAM, administered in the form of the acetic
acid ester, [14C]MAMOAc, to 14CO2,and the rates and extents
of metabolism were virtually identical. Determination of 06-methylguanine and 7-methylguanine in liver DNA 6 and 24 hr after
MAMOAc (25 mg/kg) administration showed that the levels of
DNA methylation induced by the carcinogen were not signifi
cantly different in the two strains, indicating that both are capable
of the metabolic activation of MAM to methylating species.
Pyrazole, a potent inhibitor of ADH, inhibited MAM metabolism
as well as liver DNA methylation in the ADH(+) strain; however
similar inhibition of these processes also occurred in the ADH(-)
strain. 3-Methylpyrazole, a weak or noninhibitor of ADH, also
decreased the levels of MAM metabolism in both the ADH(+)
and the ADH(-) strains. From these results, we conclude that
ADH is not obligatory either in the metabolism or in the metabolic
activation of MAM. As a possible alternative to ADH, liver microsomes were examined for their ability to metabolize MAM. In the
presence of a NADPH-generating system, liver microsomes from
both strains converted [14C]MAM to 14CH3OH and 14CH20, al
though liver microsomes from the ADH(-) strain were more
active in this respect. The microsomal metabolism was sensitive
to inhibition by CO as well as to inhibition by pyrazole and 3methylpyrazole.
INTRODUCTION
MAM,4
N—CH2OH,
I
O
1Supported in part by Grant CA 31012 from the National Cancer Institute, NIH,
Bethesda, MD.
2 This paper is dedicated to the memory of Dr. Silvio Emerich Fiala, January 1,
1911 to December 4,1981.
3To whom requests for reprints should be addressed.
4The abbreviations used are: MAM, methylazoxymethanol; MAMOAc, methylazoxymethyl acetate; ADH, alcohol dehydrogenase (alcohol:NAD* oxidoreductase, EC 1.1.1.1); ADH(+), Peromyscus maniculatus with normal ADH tissue levels;
ADH(-), Peromyscus maniculatus genetically deficient in ADH; HPLC, high-perform
ance liquid chromatography.
Received September 6,1983; accepted April 6,1984.
O
are powerful carcinogens in rodents (7, 19-21) as well as in
certain nonhuman primates (34). Because of its greater chemical
stability, MAMOAc is the preferred agent in experimental carcinogenesis, but the biological effects of the 2 compounds are
equivalent, since the blood and various tissues contain deacetylases which effect a rapid hydrolysis of the ester to the alcohol
(39). In vivo, MAM is also generated through the enzymatic
hydrolysis of the naturally occurring carcinogen, cycasin (18, 21 ,
25), as well as through the metabolic activation of 1,2-dimethylhydrazine by way of the intermediates azomethane and azoxymethane(8, 11, 13,26).
The target organs of the related carcinogens MAM, MAMOAc,
cycasin, azoxymethane, and 1,2-dimethylhydrazine are primarily
the large and small intestines, and secondarily, the liver and
kidney (reviewed in Ref. 37). The initiating event for tumor
production by these compounds is believed to be the aberrant
alkylation of DNA through the generation of the highly reactive
methyldiazonium ion from MAM (8, 28). While MAM sponta
neously decomposes, at temperatures above 0°,to the methyl
diazonium ion, "OH and formaldehyde, with a f1/2between 2.8
and 18.6 hr, depending on the conditions (10, 12, 28), it may be
assumed that this spontaneous process would not alone account
for the rather narrow range of organotropism of the carcinogen,
since it could take place in all tissues with equal facility. In 1973,
Schoental (33) proposed that the enzyme ADH may catalyze the
oxidation of MAM to a more reactive metabolite, methylazoxyformaldehyde. Evidence in apparent support of ADH in the
activation of MAM was provided by Zedeck ef a/. (10, 29, 39).
Thus, when MAM was incubated with horse liver ADH and NAD+,
a species capable of methylating the acetate anión to methyl
acetate was produced, and this process could be blocked by
pyrazole (10). Pyrazole, a classic inhibitor of ADH (36), also
prevented MAMOAc-induced lethality in rats (39), and altered
the organotropism of the carcinogen (29). However, it is abun
dantly clear that ADH is not the only enzyme system capable of
catalyzing the oxidation of various alcohols (5), and that pyrazole
can affect the activity of enzymes other than ADH (9, 14, 23,
32). Pyrazole and 4-methylpyrazole also act as scavengers of
hydroxyl radicals which have been shown to effect the oxidation
of ethanol in vitro (6, 38). In the present work, we address the
question of whether ADH is obligatory in the metabolism of
MAM. The experimental model, consisting of deer mouse strains
positive and negative for ADH, is one that had been developed
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2885
E. S. Fiala et al.
by Burnett and Felder (2-4) for the study of non-ADH-mediated
pathways of ethanol metabolism.
MATERIALS AND METHODS
Chemicals. MAMOAc was obtained from Ash Stevens, Detroit, Ml.
[14C]MAMOAc was synthesized using 1,2-[metf)y/-14C]dimethylhydrazine
dihydrochloride from New England Nuclear, Boston, MA, by a modifica
tion of the method of Horisberger and Matsumoto (17). The modification
consisted of replacing the vacuum distillation, used in the final step of
the original procedure, by purification with liquid chromatography. For
this purpose, the crude product, in 2 ml CCI«,was applied to an EM Size
B silica gel column (EM Laboratories, Elmsford, NY) equilibrated with nhexane:acetone, 95:5 (v/v), at a flow rate of 2 ml/min, and 9.5-ml fractions
were collected. Unreacted A/,A/'-[mef/7y/-14C]azoxymethane appeared in
Fractions 135 to 150, and [14C]MAMOAc appeared in Fractions 160 to
175. The latter fractions were pooled, and solvent was removed by using
a Kontes K-283500 concentrator. The radiochemical purity of the product
was >99% by HPLC, using both reverse-phase (2 Cie-^Bondapak col
umns in series eluted with methanol:H2O, 7.5:92.5, v/v), and normal
phase (2 fiPorasil columns in series eluted with n-hexane:ethanol, 98:2,
v/v. The yield of [14C]MAMOAc was approximately 10%; the specific
activity was 0.585 mCi/mmol. In several preliminary trial synthetic runs
with unlabeled precursors, high yields (>90%) up to the stage of the
synthesis of azoxymethane were obtained. However, in our hands, the
yields of MAMOAc were quite variable, which we ascribe to the incom
plete bromination of azoxymethane by W-bromosuccinimide. For more
consistent results, the use of A/-bromosuccinimide in amounts 30 to 50%
in excess of those specified by Horisburger and Matsumoto is suggested.
For use as substrate in in vitro assays. [14C]MAM was prepared by
incubating 50 n\ of ["CJMAMOAc in 1 ml of 0.1 M sodium phosphate
buffer, pH 7.5, initially with 25 n\ of a 10-mg/ml suspension of porcine
liver esterase (type I; Sigma Chemical Co., St. Louis, MO), with stirring
at 25°.The pH of the incubation mixture was maintained (15) by periodic
additions of 0.1 N NaOH via a microsyringe. After 40 min, 25 n\ of enzyme
suspension were again added, the incubation was continued for 30 min,
and then terminated by placing on ice. To purify the [14C]MAM from
protein, unhydrolyzed
[14C]MAMOAc, and decomposition
products
14CH2O and 14CH3OH, 200- to 500-/ul aliquots of the mixture were
submitted to HPLC, using a Whatman Magnum ODS column eluted with
H2O at 2 ml/min. Fractions containing MAM were stored at -20° until
used.
Pyrazole was obtained from Eastman Chemical Co., and 3-methylpyrazole was obtained from Aldrich Chemical Co. NADP*, glucose 6phosphate and glucose-6-phosphate dehydrogenase were obtained from
Sigma Chemical Co., as was 7-methylguanine. O6-Methylguanine stand
ards were kindly provided by Dr. A. Pegg and also by Dr. F. L. Chung.
Animals. The animals used in these studies were specially derived
strains of the deer mouse, Peromyscus maniculatus, originally developed
at the Department of Biology, University of South Carolina, Columbia,
SC. One strain, designated as ADH(+), is characterized by a normal
complement of ADH. The other, designated as ADH(-), is characterized
by a complete absence of the enzyme in the liver, as well as in other
organs (2-4). Upon arrival at the Naylor Dana Institute, the deer mice
were marked by ear punch, and the ADH status of randomly selected
animals from each group was verified in liver, kidney, lung, and colon
cytosols. Integrity of the ADH(+) and ADH(-) phenotypes was maintained
by selective breeding, with periodic monitoring of the ADH status. The
animals were housed in solid-bottom polycarbonate cages containing
hardwood chip bedding, with free access to NIH-07 diet and water. The
environment of the animal rooms was controlled as to temperature [21
±1°(S.D.), relative humidity (50 ±10%), and light-dark cycles (12 hr)].
Metabolism in Vivo. Weight-matched male ADH(+) or ADH(-) deer
mice were given injections s.c. of 25 mg [14C]MAMOAc/kg dissolved in
0.9% NaCI solution, and immediately
2886
placed, in groups of 3, in glass
metabolism cages. Air, dried and freed of CO2, was drawn through the
cages at 150 ml/min. Determination of exhaled 14CO2was performed as
previously described (13, 14). At the end of 24 hr, the mice were
sacrificed, and livers were quickly removed for ADH assays and the
isolation of DNA.
Assay of 7-Methylguanine and O'-Methylguanine in Liver DNA. At
various periods following the administration of 14C-labeled or unlabeled
MAMOAc, the livers of 3 ADH(+) or ADH(-) deer mice were pooled,
rinsed with ice-cold 0.9% NaCI solution, and stored at -70°. DNA was
isolated by the procedure described by Margison and Kleihues (24). As
published, the procedure contains a typographical error; 0.3 M, rather
than 3.0 M, sodium acetate is to be used in the step involving the
extraction of crude DNA.
Purified liver DNA was hydrolyzed in 0.1 N HCI at 37°for 18 hr. The
samples were then neutralized with dilute NaOH solution, and taken to
dryness with a Savant Instruments Speed-Vac centrifugal concentrator.
The samples were dissolved in 0.1 ml of 0.1 M ammonium phosphate
buffer, pH 2.0, and submitted to HPLC, using a 0.46 x 25-cm Whatman
Partisi) SCX column eluted with the same buffer at 2.0 ml/min. The
column effluent was monitored at 295 nm, using a Kratos Model SF770
variable wavelength UV detector for the quantitation of guanine. In turn,
the effluent from the UV detector was routed to a Perkin Elmer Model
650-1 OS fluorescence spectrophotometer (excitation, 295 nm; emission,
370 nm) for the quantitation of O6-methylguanine and 7-methylguanine.
In cases when radioactive carcinogen had been used, fractions of 1 ml
were collected and 14Cwas determined by liquid scintillation counting.
Enzyme Assays. Deer mouse livers, kidneys, lungs, or colon mucosa
were homogenized in 2 weight volumes of ice-cold 0.25 M sucrose, 0.01
M in sodium phosphate, pH 7.5. The homogenate was centrifuged at
9,000 x g for 20 min. Cytosol and microsomal fractions were obtained
by centrifuging the resulting supernatant at 100,000 x g for 60 min.
ADH was assayed in the cytosol fractions by the reverse reaction (2)
in an assay mixture consisting of 3.3 mw sodium pyrophosphate, pH
6.5, 0.14 mM NADH, and 2 mw acetaldehyde. The oxidation of NADH to
NAD ' was monitored by the decrease in optical density at 340 nm at
25°.
For assays of MAM metabolism by liver microsomes, the microsomal
fraction was weighed and resuspended in 3 weight volumes of 0.1 M
sodium phosphate buffer, pH 7.O. Incubation media contained in 0.5 ml:
100 /iinol sodium phosphate buffer, pH 7.0; 1.75 //mol glucose 6phosphate; 5 units of glucose-6-phosphate dehydrogenase; 0.75 ¿tmol
NADP+; 1.75 ^mol MgCI2; 50 nmol [14C]MAM, and approximately 0.5 mg
(protein) liver microsomes. Incubations were performed in 7-ml glass
scintillation vials maintained at 37°in a water bath with linear shaking at
97 cycles/min. Incubations were terminated by placing the vials on ice
and adding 0.5 ml of cold methanol. Aliquots of the supernatants obtained
after centrifugatton at 0°were submitted to HPLC, using 2 Hamilton
PRP-1 columns (0.41 x 15 cm) in series, eluted with H20 at 1 ml/min.
The absorbance of the effluent was monitored at 215 nm, and fractions
of 0.5 ml were collected for the determination of radioactivity.
For assays of MAMOAc deacetylase activity, deer mice were decapi
tated and blood was collected in heparinized tubes. Livers or kidneys
were homogenized in 4 or 8 times, respectively, their weight volumes of
0.1 M potassium phosphate, pH 7.3, and filtered through gauze. Aliquots
of the tissue homogenates or blood, total protein concentration approx
imately 3 mg/ml, were incubated at 37° in 1 ml of 0.1 M potassium
phosphate buffer, pH 7.3, with 0.1 M MAMOAc (12). After 10 min of
incubation, 3 ml of ice-cold methanol were added, and the mixtures were
centrifuged at 0°. MAM, liberated from MAMOAc, was determined in
aliquots of the supernatants by HPLC, using a C,a-«¿Bondapak
column
eluted with methanol:H2O, 5:95 (v/v), and monitoring the UV absorption
of the effluent at 215 nm. A relationship between the peak height of
eluted MAM and concentration was established, using the molar absorp
tivity value of 8710, as given by Kobayashi and Matsumoto (18). Protein
was determined by the Lowry method. Control incubations were carried
out, either without MAMOAc or without tissue or blood preparations.
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VOL. 44
Methylazoxymethanol Acetate Metabolism
metabolized to 14CO2in 2 hr, and 67 ±1.5% was metabolized
RESULTS
In Vivo Metabolism.
The rates of metabolism of
[14C]MAMOAc, administered as a single s.c. dose of 25 mg/kg,
to exhaled 14CO2by ADH(+) and ADH(-) deer mice are shown
in 24 hr. These results show that ADH is not obligatory in the
metabolism of MAM.
The same animals used in the above metabolism study were
sacrificed 24 hr after the [14C]MAMOAc administration, and ADH
in Chart 1. The metabolism of the carcinogen is rapid in the
ADH(+) strain; approximately 47 ±2% (S.D.) of the dose was
exhaled as 14CO2in 2 hr, and 64 ±3% was exhaled within 24
hr. Comparison with the ADH(-) strain indicates that metabolism
in the latter occurs just as rapidly: 47 ±2% of the dose was
was assayed in livers, kidneys, lungs, and colon mucosa. The
results of these assays, shown in Table 1, confirmed the ADH
status of the deer mice used. No detectable ADH activity was
found in the organs of the ADH(-) strain, while varying levels of
ADH were found in the homologous organs of the ADH(+) strain.
The levels of the enzyme in the ADH(+) strain were in the order
of liver > kidney > colon > lung, when calculated per mg protein.
Assays of ADH in either deer mouse strain without previous
administration of MAMOAc gave results not significantly different
from those obtained with MAMOAc-treated animals.
Effects of Pyrazoles. Pyrazole is a potent inhibitor of ADH
both in vivo and in vitro (22, 36). Pretreatment of rats with
pyrazole had been reported to decrease the acute toxicity of
MAMOAc (15), and also to alter the organotropism of the carcin
ogen (29). It was therefore of obvious interest to examine the
effects of this compound on the metabolism of [14C]MAMOAc.
As shown in Chart 1, pretreatment with 120 mg pyrazole/kg
(i.p.) 2 hr prior to the administration of [14C]MAMOAc (s.c.)
2
4
6
24
HOURS
2
4
6
24
HOURS
Chart 1. Time course of in vivo metabolism of [14C]MAMOActo exhaled "CDs
by ADH(+) and ADH(-) deer mice, and the effects of pretreatment with pyrazole
or 3-methylpyrazole. All mice received [14C]MAMOAc,25 mg/kg, s.c., at Time 0.
A, no pretreatment; •,pyrazole, 120 mg/kg, i.p., at -2 hr; O, 3-methylpyrazole,
140 mg/kg, i.p., at -2 hr; A, 3-methylpyrazole, 140 mg/kg, i.p., at -15 min. Points,
means of replicate determinations, each using 3 mice per metabolism chamber.
Bars, S.D. (some omitted for clarity). Numbers in parentheses, number of times
each experiment was performed.
Table 1
ADH activity in organs from MAMOAc-treatedand untreated deer mice
protein)8Liver
ADH activity (nmol NADH oxidized/min/mg
treated6ADH(+)
ADH(-)175±25c(4)d
0(2)
±36(5)
89 ± 4 (2)
0(2)
112 ±32 (5)
0(2)
Kidneys
0(2)
4± 5(2)
2± 1 (3)
0(2)
Lung
69 ± 4(3)ADH(-)0(2)
0(3)
0(2)UntreatedADH(+)166
Colon mucosaMAMOAc64 ± 3 (3)
8 ADH activity was determined as described in "Materials and Methods."
b Assays were performed 24 hr after treatment with MAMOAc (25 mg/kg, s.c.).
c Mean ±S.D.
" Numbers in parentheses, number of separate determinations, each performed
using cytosols from organs of individual animals.
caused an 89% decrease in the in vivo conversion of the carcin
ogen to 14CO2in the ADH(+) strain 2 hr after dosing with [14C]MAMOAc, and a 71 % decrease at 6 hr. The same treatment in
the ADH(-) strain caused similar decreases: 88% at 2 hr and
67% at 6 hr.
Substitution at the 3-position of pyrazole substantially de
creases its activity as an ADH inhibitor. Thus, the Ki of 3methylpyrazole, with respect to ethanol, is some 600 times
higher than that of pyrazole, using horse liver ADH (37). If ADH
were involved in the metabolism of MAM, little or no inhibition by
3-methylpyrazole pretreatment would be expected. However, as
shown in Chart 1, pretreatment of deer mice with 140 mg of 3methylpyrazole/kg, ¡.p.,2 hr prior to the administration of [14C]MAMOAc caused an 84% inhibition of exhaled 14C02 in the
ADH(-t-) strain, and a 92% inhibition in the ADH(-) strain at 2 hr
after administration of the carcinogen. At 6 hr following [14C]MAMOAc, a 39% inhibition occurred in the ADH(+) strain, and a
39% inhibition in the ADH(-) strain. Since a greater inhibition
due to 3-methylpyrazole was occurring during the initial phase
of the study (i.e., 0 to 2 hr) than during the later phase (i.e., 2 to
6 hr), it appeared likely that the effective level of the inhibitor in
Table 2
O"-Methylguanine and 7-methylguaninelevels in liver DNA isolated from ADH(+) orADH(-) deer mice 6 and
24 hr after MAMOAc (25 mg/kg, s.c.) treatment with and without pyrazole (120 mg/kg, i.p.) pretreatment 2 hr
prior to MAMOAc
/imol O6-methylguanine/molguanine
fimol 7-methylguanine/molguanine
Deer mouse strain and
24 hr
24 hr
6hr
6hr
pretreatment
ADH(+)
No pretreatment
pretreatmentADH(-)
Pyrazole
±46a (A)"
(E)402
119 ±46
NDC61
68 ±2361(C)
165(F)6717
1592±
8 ±385 (D)
ND3422
±1007(I)
±577 (J)
3 ± 73 (H)
±92 (G)
No pretreatment
2306 ±1043 (L)271
ND
ND61
Pyrazole pretreatment522 229 ±64 (K)467±110(B)
8 Mean ±S.D. from 3 to 4 separate determinations,each performed on DNA isolated from the pooled livers
of 3 deer mice.
* Significant differences between the groups (f test) are as follows: A versus E, p < 0.001; G versus K, l
versus L, and I versus J, p < 0.005; C versus F, and C versus D, p < 0.025.
c ND, not determined.
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2887
E. S. Fiala et al.
the animals was decreasing. To examine whether this were the
case, sets of ADH(+) and ADH(-) deer mice were pretreated
with 3-methylpyrazole 15 min prior to the administration of
[14C]MAMOAc, and exhaled 14CO2was again determined. The
results, also shown in Chart 1, indicate that while the inhibition
of exhaled 14CO2 during the first 2 or 3 hr was the same
regardless of the time of pretreatment, after this period, inhibition
was more persistent in the 15-min pretreatment groups than in
the 2-hr pretreatment groups. This is what would be expected if
the effective levels of 3-methylpyrazole in the animals were
decreasing with time due to metabolism and/or excretion. In
terms of inhibition of MAM metabolism, differences between the
15-min and 2-hr 3-methylpyrazole pretreatments are statistically
significant (p < 0.02) at the 6- and 7-hr time points in the ADH(-)
mice. The analogous values obtained with the ADH(+) mice
show a similar trend; however, the differences are not statistically
significant (p > 0.05).
DNA Methylation. The rate of conversion of a carcinogen to
CO2 may not necessarily be correlated with the rate of its
metabolic activation. To obtain an indication of the metabolic
activation of MAM in the 2 deer mouse strains, liver DNAs were
isolated 6 and 24 hr after treatment with 25 mg MAMOAc/kg,
and the levels of O8-methylguanine and 7-methylguanine were
determined. The results, shown in Table 2, indicate that the
degree of DNA methylation in the ADH(+) strain was not signifi
cantly different from that in the ADH(-) strain, either at the 6-hr
or 24-hr time point. In both strains the levels of 7-methylguanine
were higher at 6 hr after the administration of MAMOAC than
they were at 24 hr after the administration, indicating rapid loss
of this methylated base from liver DNA. These results are similar
to those obtained for rat liver DNA after 1,2-dimethylhydrazine
cient to hydrolyze virtually all of the MAMOAc administered
(approximately 2.8 /¿mol/mouse)by the liver (approximately 0.8
g/15-g mouse) in less than a minute, even if contribution to the
hydrolysis by other organs were neglected. Thus, inhibition of
MAMOAc metabolism by the pyrazoles at this locus is excluded.
In Vitro Metabolism. Among pathways of alcohol metabolism
alternative to ADH, the microsomal ethanol-oxidizing system has
been the most extensively studied (5, 6, 35, 38). We therefore
examined deer mouse liver microsomes for their ability to metab
olize MAM.
In the presence of a NADPH-generating system, deer mouse
liver microsomes were found to metabolize [14C]MAM to 2 polar
species which eluted on reverse-phase HPLC (2 PRP-1 columns
in series eluted with H20) near the void volume.
of elution volumes with 14C-labeled standards,
were tentatively identified as 14CH20, eluting
14CH3OH, eluting at approximately 4 ml. These
were obtained as a result of the spontaneous
[14C]MAM (Chart 2).
guanine and 7-methylguanine in liver DNA were found, compared
to mice not pretreated with pyrazole (Table 2). Similar inhibition,
by pyrazole, of methylation of rat colon DNA following 1,2dimethylhydrazine treatment had been noted by Bull ef al. (1).
The foregoing results indicate that the ADH status of the deer
mice is not a factor, either in the overall metabolism, or in the
metabolic activation of MAM to a species methylating DNA.
Possible Effects of Pyrazoles on Deacetylation of
MAMOAc. Although unlikely, it nevertheless seemed possible
that the decreased rates of [14C]MAMOAc metabolism and liver
DNA methylation following pyrazole of 3-methylpyrazole pre
treatment in both strains of deer mice might be due to the
inhibition of the hydrolysis of the acetate ester to MAM. To
examine this possibility, the deacetylation of MAMOAc was
determined in vitro, using either blood or homogenates of livers
or kidneys of nonpretreated animals incubated with and without
pyrazole or 3-methylpyrazole (1 rnw), as well as in similar prep
arations obtained from animals pretreated with pyrazole (120
mg/kg, i.p.) or 3-methylpyrazole (140 mg/kg, i.p.) 2 hr prior to
sacrifice. While considerable variability in experimental results
was obtained (Table 3), due perhaps in part to interanimal
differences in enzyme levels, no consistent effects due to pre
treatments with either of the 2 pyrazoles, or to their inclusion in
the deacetylase assay system, were noted. Moreover, in all
cases, the levels of deacetylase assayed in vitro, albeit under
conditions of enzyme saturation with substrate, would be suffi
2888
breakdown
of
Tabte3
Lack of inhibition of MAMOAc deacetylases by pyrazole or 3-methylpyrazole
treatment
Heparinized whole Wood or tissue homogenates from deer mice, either untreated
or treated with pyrazole (120 mg/kg, i.p.) or 3-methylpyrazote (140 mg/kg, i.p.) 2
hr prior to sacrifice, were incubated at 37°with MAMOAc (0.1 M) in 0.1 M potassium
phosphate buffer, pH 7.3. Blood or tissue homogenates from untreated deer mice
were also incubated with the addition of either pyrazole or 3 methylpyrazole (1 RIM)
to the system. Incubations were terminated by the addition of 3 volumes of
methanol. After centrifugation, MAM was determined in the supematants by HPLC.
MAMOAc deacetylase activity (^mol MAM formed/mg protein)
(no addition
system)Pyrazote0.77
to
treatment)ADH(+)
Addition to system (no
treatment (16, 31).
When pyrazole was given to both the ADH(+) and ADH(-)
deer mice at 120 mg/kg, ¡.p.,2 hr prior to MAMOAC, and the
mice were sacrificed 6 hr later, decreased levels of 06-methyl-
By comparison
these species
at 3 ml, and
same products
pyrazole0.80
pyrazote0.85
Liver
Kidney
BloodADH(-)
0.47
0.050.83
0.54
0.050.78
0.42
0.040.88
0.61
0.050.88
0.31
0.050.83
Liver
Kidney
0.43
0.40
0.59
0.36
0.29
BloodNone0.65 0.04Pyrazole0.64
0.033-Methyl-0.04Treatment 0.063-Methyl-0.05
2000-1
2OOO-
OD
150°-<(£
B
15OO-
U.g
1000-
1000lÃ-lol
50°-ûA_•i\
2
4
6
500-
8
10
8
10
ELUTION VOLUME, mi
Chart 2. HPLC profiles of products of [14C]MAM decomposition and metabo
lism. ["CJMAM, 0.1 mm, was incubated as described in "Materials and Methods,"
with a NADPH-generating system, at pH 7.0, without (A) or with (B) liver microsomes
from ADH(-) deer mice (approximately 0.5 mg/ml) for 15 min at 37°.After chilling
to 0°,the addition of methanol and centrifugation, supematants were analyzed by
HPLC (2 Hamilton PRP-1 columns in series, eluted with water). Radioactivity eluting
at 2.5 ml represents '4CH2O; at 3.5 ml, 14CH3OH; at 6 ml, [14C]MAM. Qualitatively
similar profiles were obtained with liver microsomes from ADH(+) mice.
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VOL. 44
Methylazoxymethanol Acetate Metabolism
The degree of [14C]MAM metabolism was calculated by sub
tracting the amount of radioactivity in these 2 peaks found using
heat-denatured (90°,10 min) microsomes from that found using
non-heat-denatured microsomes incubated in otherwise identical
systems for the same periods of time.
Chart 3 compares the rates of metabolism of [14C]MAM, using
liver microsomes from ADH(+) and ADH(-) deer mice. In incu
bation systems containing 0.5 mg microsomal protein, liver mi
crosomes from ADH(-) deer mice metabolized [14C]MAM some
Table 4
Factors influencing the in vitro metabolism of [Ì4C]MAMby deer mouse liver
microsomes
[14C]MAM, 0.1 rriM, was incubated for 15 min at 37°with liver microsomes (0.5
mg) obtained from the pooled livers of 3 ADH(+) or ADH(-) deer mice in 0.5 ml of
medium containing 100 ^mol sodium phosphate buffer, pH 7.0, 1.75 »imolglucose
6-phosphate, 5 units of glucose-6-phosphate dehydrogenase, 4.5 jimol nicotinamide, 0.75 fimol NADP+, and 1.75 nmol MgCI2 (complete system). Omissions or
additions to the system were made as indicated. Products of the reaction were
determined by HPLC as described in "Materials and Methods."
% of activity
ADH(+) microsomes
30 times faster than the rate of spontaneous breakdown; liver
microsomes from the ADH(+) mice metabolized [14C]MAM ap
ADH(-) microsomes
Complete
systemWithout
NADPC0:02
atmospherePlus
ITIM)Plus
pyrazole (0.1
mM)Plus
pyrazole (1 .0
HIM)Plus
3-methylpyrazole (0.1
mM)Heated
3-methylpyrazole (1 .0
microsomes1000038619501000047112100
proximately 18 times faster than the spontaneous rate.
The rates of [14C]MAM metabolism were linear functions of
the concentration of microsomal protein, up to approximately 1
mg of incubation mixture/ml (Chart 4); at higher microsomal
protein concentrations, only a slight further increase was ob
served.
As shown in Table 4, omission of NADP+ from the incubation
systems resulted in complete loss of activity toward MAM;
likewise, incubation under an atmosphere of carbon monox-
used. It is of considerable interest that, as in the case of metab
olism of [14C]MAMOAc in vivo, both pyrazole and 3-methylpyra
ide:oxygen, 80:20, resulted in complete inhibition. No activity
was seen when heat-denatured (90°,10 min) microsomes were
zole are effective inhibitors of the reaction in vitro. At a concen
tration of 10~4 M pyrazole, a 62% inhibition of the ADH(+) and a
53% inhibition of the ADH(-) systems was observed. Similarly,
at the same concentrations of 3-methylpyrazole, an 81% inhibi
tion of the ADH(+) system and a 79% inhibition of the ADH(-)
system occurred. Increasing the concentrations of the 2 pyrazoles to 10~3 M resulted in greater than 89% inhibition in all
cases.
DISCUSSION
The results of our in vivo studies show that [14C]MAMOAc is
metabolized to 14C02 equally rapidly by deer mice with normal
2
<
ADH levels as by deer mice totally lacking the enzyme. Both
strains metabolize 50% of the dose to CO2 in approximately 2.5
hr. Since the spontaneous decomposition of MAM occurs with
a f1/2of 14 hr at 37°and physiological pH (10), the necessary
2-
10
20
INCUBATION
TIME.
30
MIN
Chart 3. Rates of metabolism of [I4C]MAM by liver microsomes from ADH(+)
and ADH(-) deer mice. [14C]MAM, 0.1 mw, was incubated at 37°for various times
in the presence of liver microsomes, phosphate buffer, pH 7.0, and an NADPHgenerating system. The products of the reaction were determined as described in
'Materials and Methods." Points, means of separate determinations, using pooled
liver microsomes from groups of 2 to 3 mice. Bars. S.D. Numbers in parentheses,
number of groups of mice used to obtain that value. All values have been corrected
for the spontaneous rate of decomposition (0.04 nmol MAM/min at 37°)determined
from identical assays carried out with heat-denatured microsomes.
conclusion from these experiments is that enzymes other than
ADH are responsible for MAM metabolism, at least in the ADH(-)
strain. That such metabolism is associated with the production
of a reactive species from MAM is demonstrated by the finding
of approximately the same extents of liver DNA methylation in
the ADH(+) and the ADH(-) strains, as reflected in the liver DNA
O6-methylguanine and 7-methylguanine levels.
Pretreatment with pyrazole inhibited the metabolism of
MAMOAc, not only in the ADH(+), but also in the ADH(-) strains,
by 88 to 89% at the 2-hr time point after MAMOAc administra
tion. The lack of inhibition, by pyrazole and its 3-methyl derivative,
E
If)
321-
1.O
2.0
PROTEIN CONCENTRATION
mg/ml
Chart 4. Dependence of in vitro metabolism of [UC]MAM on concentration
of
microsomal protein. Incubation (15 min) and analysis of products were performed
as described in 'Materials and Methods.*
of deacetylases in deer mouse tissues (Table 3) excludes an
effect at this locus. Also, the effect appears to be due to the
actual inhibition of MAM metabolism, rather than to the inhibition
of oxidation of the MAM metabolites and/or breakdown prod
ucts, CH3OH and CH2O, since Phillips ef al. (30) have found that
pyrazole pretreatment has no effect on the in vivo metabolism
of CH3OH (and therefore also of CH2O) to CO2. Moreover, as
shown in Table 2, pyrazole also inhibited the methylation of liver
DNA in both strains. This indicates that the enzyme(s) catalyzing
the metabolism of MAM, already demonstrated to be different
from ADH, nevertheless resemble(s) ADH in terms of pyrazole
sensitivity. Previous conclusions as to the relevance of ADH in
JULY 1984
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2889
E. S. Fiala et al.
thèin vivo metabolism of MAM have relied on the observations
that pyrazole, a known inhibitor of ADH, decreases the acute
toxicity of MAMOAc in rats, and alters the organotropism of the
carcinogen (15, 29). The present results add to the evidence
already in the literature that pyrazole can affect enzymes other
than ADH. Thus, pyrazole is known to bind to microsomal
hemoproteins, producing a type 2 difference spectrum, and
inhibits a variety of microsomal drug-metabolizing activities (23,
32). The inhibition of the metabolic activation of azoxymethane
to MAM by pyrazole, both in vivo and by a liver microsomal
system in vitro, has previously been demonstrated (14), and
pyrazole has been shown by Moriya et al. (27) to inhibit the
carcinogenicity of azoxymethane and 1,2-dimethylhydrazine in
rats. Both pyrazole and its 4-methyl derivative have been shown
to inhibit the hydroxyl radical-mediated oxidation of ethanol by
the ascorbate:iron:EDTA and xanthine:xanthine oxidase sys
tems, and by rat liver microsomes (6). In addition to its inhibitory
effects, pyrazole has been described to act as an inducer of
microsomal enzymes, in particular of dimethylnitrosamine demethylase (9). Clearly, as has been emphasized before (6, 23),
when a biological effect is observed due to the administration of
pyrazole, it cannot be ascribed solely to the inhibition of ADH.
The effects of 3-methylpyrazole, a weak or non-inhibitor of
ADH, on [14C]MAMOAc metabolism in both strains of deer mice
resemble those of pyrazole, especially during the first hr or so
after the administration of the carcinogen. This suggests that
the enzyme(s) involved in the metabolism of MAM in the ADH(-)
strains are also operative in the ADH(-i-) strain. After the first hr,
the degree of inhibition is greater in the ADH(-) mice than in the
ADH(+) mice. This effect may reflect a contribution of ADH to
the metabolism of MAM in the mouse strain possessing this
enzyme, although additional differences between the 2 strains in
terms of disposition and metabolism of 3-methylpyrazole cannot
be ruled out. That metabolism and/or elimination of 3-methylpyr
azole plays a role in the degree of inhibition of MAM metabolism
is suggested by the greater persistence of inhibition when 3methylpyrazole is given 15 min prior to [14C]MAMOAc than when
it is given 2 hr prior to the carcinogen (Chart 1).
It is apparent from the results given in Chart 3, that liver
microsomes from the ADH(-) mice are more active in MAM
metabolism than are liver microsomes from the ADH(+) mice.
Yet, the rates of the in vivo metabolism of MAMOAc in the 2
mouse strains are not significantly different (Chart 1). Thus
microsomal metabolism of MAM may provide a pathway alter
native to ADH in the ADH(-) mouse strain, and a pathway
supplementary to ADH in the ADH(+) strain. The microsomal
enzymes involved have not as yet been fully characterized, but
the sensitivity of the MAM metabolism to CO inhibition (Table 4)
suggests the participation of some form of cytochrome P-450.
As in the case of the in vivo metabolism of MAMOAc, the liver
microsomal metabolism of MAM in vitro is inhibited by both
pyrazole and 3-methylpyrazole in both the ADH(+) and ADH(-)
deer mouse strains. In view of the findings of Cederbaum and
Beri (6), and Winston and Cederbaum (38), that the microsomal
metabolism of ethanol is mediated, at least partly, through the
production of the highly reactive hydroxyl radicals, and that
pyrazole and 4-methylpyrazole act as hydroxyl radical scaven
gers, thus inhibiting microsomal ethanol oxidation, it is tempting
to speculate that the microsomal metabolism of MAM involves
similar mechanisms. Studies to examine this possibility are being
planned.
2890
Clearly, ADH(+) and ADH(-) deer mice can be of great use in
delineating the metabolic pathways of MAM activation, without
introducing ambiguities of interpretation inherent in the In vivo
utilization of inhibitors, such as pyrazole, whose effects are not
absolutely specific for a single enzyme. However, a disadvantage
of the deer mouse model is that, due to their relative scarcity,
the carcinogenicity of MAM in these animals has not as yet been
defined. Efforts are being made in this laboratory to breed a
sufficient number of each of the deer mouse strains for such
studies. In addition, the microsomal system is being examined in
further detail, with the aim of evaluating its contribution to the
metabolism of MAM in rodent species for which the carcinogen
icity of MAMOAc is known. That the microsomal MAM-metabolizing system is present in species other than Peromyscus is
already indicated by preliminary data which show that the micro
somal fractions of both livers and colons of F344 rats are active
in MAM metabolism.5
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2891
Non-Alcohol Dehydrogenase-mediated Metabolism of
Methylazoxymethanol in the Deer Mouse, Peromyscus
maniculatus
Emerich S. Fiala, Nancy Caswell, Ock Soon Sohn, et al.
Cancer Res 1984;44:2885-2891.
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