Environmental Health Perspectives Volume
102, Supplement 6, October 1994
[Citation
in PubMed] [Related
Articles]
Cytosolic Activation of Aromatic and Heterocyclic Amines. Inhibition
by Dicoumarol and Enhancement in Viral Hepatitis B
Silvio De Flora, Carlo Bennicelli, Francesco D'Agostini, Alberto Izzotti,
and Anna Camoirano
Institute of Hygiene and Preventive Medicine, University of Genoa, Italy
Abstract
The aromatic amines 2-aminofluorene (2AF), 2-acetylaminofluorene, and
2-aminoanthracene, and the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline
(IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, and 3-amino-1-methyl-SH-pyrido[4,3-b]indole
(Trp-P-2) were activated by rat liver cytosolic fractions to form mutagenic
metabolites in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6.
In the case of the Trp-P-2, the cytosolic activation was even more potent
than the microsomal activation, which is classically ascribed to N-hydroxylation
and subsequent esterification. The cytosolic activation was a) NADPH-dependent,
b) induced by pretreatment of rats with 3-methylcholanthrene and
especially Aroclor 1254 but not by phenobarbital, and c) inhibited
by dicoumarol. The hypothesis is that, following a preliminary oxidative
step in the cytosol (pure cytosolic activation) or in microsomes via prostaglandin
H synthase (mixed microsomal-cytosolic activation), an oxidized intermediate
of amino compounds may serve as substrate for DT diaphorase activity and
bielectronically reduced to the corresponding N-hydroxyamino derivative.
Purified DT diaphorase, in the presence of either NADPH or NADH as electron
donor, produced mutagenic derivatives from IQ and Trp-P-2. An NADPH-dependent
activation of Trp-P-2 also occurred in the liver cytosol of woodchucks (Marmota
monax), but was not inhibited by dicoumarol. As previously demonstrated
with liver S-12 fractions in both humans and woodchucks, the cytosolic activation
of Trp-P-2 was enhanced in animals affected by hepatitis B virus infection.
This enhanced metabolism, which persisted even after appearance of primary
hepatocellular carcinoma in virus carriers, is likely to be ascribed to
mechanisms other than DT diaphorase induction, such as glutathione depletion.
-- Environ Health Perspect 102(Suppl 6):69-74 (1994)
Key words: aromatic amines, heterocyclic amines, metabolism, cytosol,
mutagenicity, dicoumarol, DT diaphorase, viral hepatitis
This paper was presented at the Fifth International Conference
on Carcinogenic and Mutagenic
N-Substituted Aryl Compounds held 18-21 October 1992 in Würzburg,
Germany.
This study was supported by the National Research Council
(CNR Targeted Project "Prevention and Control of Disease Factors -
FATMA").
Address correspondence to S. De Flora, Institute of Hygiene
and Preventive Medicine, University of Genoa, Via A. Pastore 1, I-16132
Genoa, Italy. Telephone 39 10 353 8500. Fax 39 10 353 8504.
Introduction
The first metabolic step in the hepatic activation of mutagenic and carcinogenic
aromatic amines (1) and of food-derived heterocyclic amines (2)
consists in the microsomal oxidation of the exocyclic amino group, which
is primarily catalyzed by cytochromes P4501A1 and especially P4501A2 (3).
The N-hydroxyamino derivatives are further esterified to form more
reactive species, [e.g., via O-sulfation (4) or O-acylation,
which takes place both in mammalian liver cytosol (5) and bacterial
cells (6 )]. An alternative pathway, especially in extrahepatic tissues,
proceeds via one-electron oxidation catalyzed by microsomal prostaglandin
H synthase (PHS) (7,8).
Less attention has been paid to the exclusive activation of these compounds
in the liver cytosol, although an activation of aromatic amines to mutagenic
metabolites has already been reported to occur in the presence of rat liver
cytosolic fractions in bacterial test systems (9-11).
We present herein the results of studies investigating the liver cytosolic
metabolism of the aromatic amines 2-aminofluorene (2AF), 2-acetylaminofluorene
(2AAF) and 2-aminoanthracene (2AA), and of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f
]quinoline (IQ), 2-amino-3,4- dimethylimidazo[4,5-f ]quinoline
(MeIQ) and 3-amino-1-methyl-SH-pyrido[4,3-b]-indole (Trp-P-2). The
cytosolic activation of all these compounds was inhibited by dicoumarol,
a rather specific inhibitor of DT diaphorase activity (12). Addition
of purified DT diaphorase, using either NADH or NADPH as electron donor,
produced mutagenic derivatives of heterocyclic amines. In addition, in agreement
with a previous study using S-12 fractions (13), the activation of
Trp-P-2 by liver S-105 fractions was enhanced in woodchucks naturally infected
with hepatitis B virus (WHV).
Materials and Methods
Chemicals and Biochemicals
Test mutagens included the heterocyclic amines Trp-P-2, IQ, and MeIQ
(gifts of T Sugimura and K Wakabayashi, National Cancer Center Research
Institute, Tokyo, Japan), and the aromatic amines 2AF (Ega-Chemie KG, Steinheim/Albuch,
Germany), 2AAF and 2AA (both from Sigma Chemical Co., St. Louis, MO). All
test mutagens were dissolved and diluted in dimethylsulfoxide (DMSO). Dicoumarol
(Sigma) was dissolved in 0.01 N NaOH. DT diaphorase (EC 1.6.99.2), purified
from the liver cytosol of 3-methylcholanthrene-treated rats (14),
was a gift from L Ernster, C Lind, and J Segura Aguilar (Arrhenius Laboratory,
University of Stockholm, Sweden). Its molecular activity, as assayed with
menadione as substrate (15), was 72,500 mole/mole FAD/min.
Animals
Four groups (five animals each) of male adult Sprague-Dawley rats (Morini
strain) were either untreated or treated with one of the following inducers
(all of them diluted in corn oil): phenobarbital (Merck AG, Darmstadt, Germany)
3 ip injections of 60 mg/kg during the 3 days before killing, 3-methylcholanthrene
(Fluka AG, Buchs, Switzerland) single ip injection of 80 mg/kg 24 hr before
killing, or Aroclor 1254 (Monsanto Co., St. Louis, MO) single ip injection
of 500 mg/kg 5 days before killing.
The livers of 17 woodchucks (Marmota monax), either uninfected
or infected with WHV or additionally carrying primary hepatocellular carcinoma
(PHC), were kindly supplied by I Millman (Fox Chase Cancer Center, Institute
for Cancer Research, Philadelphia, PA).
Preparation of Liver Subcellular Fractions
Liver preparations were obtained as previously described (16).
They included: a) whole cell homogenates, obtained by homogenizing,
in a Potter-Elvehjem apparatus, minced livers in a 50 mM Tris-0.25 M sucrose
solution, pH 7.4 (3 ml/g wet tissue); b) S-12 fractions, i.e., supernatants
obtained by twice centrifuging cell homogenates for 20 min at 12,000g;
c) S-105 or cytosolic fractions, i.e., supernatants obtained by centrifuging
S-12 fractions for 1 hr at 105,000g; and d) microsomal fractions,
i.e., the corresponding pellets, washed once and resuspended in a 50 mM
Tris 0.1 M EDTA solution, pH 7.4, supplemented with 20% glycerol (0.5 mg/g
of original tissue). All the cell preparations were divided into small aliquots
and immediately stored at -80°C until use. For use in mutagenicity assays,
liver preparations were thawed and incorporated, in varying amounts, into
S-9 mix, i.e., an NADPH-generating system composed of 8 mM MgCl2,
33 mM KCl, 5 mM G6P, 4 mM NADP+ and 100 mM sodium phosphate,
pH 7.4; in assays with microsomes this mix was supplemented with yeast G6PD
(8 IU/ml).
Mutagenicity Assays
The mutagenicity of test compounds in the presence of the above described
metabolic systems, at the doses indicated under Results, was evaluated in
the Salmonella reversion test, according to the plate incorporation procedure
(17), using the S. typhimurium strains TA98 (gift of BN Ames,
University of California, Berkeley, CA) and its nitroreductase- or O-acetyltransferase-deficient
derivatives TA98NR and TA98/1,8-DNP6 (gifts of H.S. Rosenkranz,
University of Pittsburgh, PA). Briefly, two consecutive preincubation steps
were performed, the first (10 min at 37°C) involving the mixture of
500 µl either of metabolic systems or its control (lacking liver preparations
and/or cofactors) with 100 µl of either dicoumarol or its solvent,
the second (30 min at 37°C) involving incubation with 100 µl of
either test mutagens or their controls (DMSO) before plating in top agar
with the appropriate Salmonella strain. All the assays were performed in
triplicate plates.
Results
The mutagenicity assay of the heterocyclic amines Trp-P-2, IQ, and MeIQ
in strain TA98 of S. typhimurium, in the presence of varying amounts
of liver subcellular fractions from Aroclor-treated rats (Figure 1), showed
that the activation by reconstituted cytosolic plus microsomal fractions
is even greater than that produced by the original postmitochondrial fractions.
In addition, cytosolic fractions were as active (IQ and MeIQ) or even more
active (Trp-P-2) than the corresponding microsomal fractions, at equivalent
weight of liver, in activating these heterocyclic amines. The addition of
dicoumarol resulted in an evident decrease of the mutagenicity of all three
compounds, and especially of Trp-P-2, in the presence of S-12, S-105, or
S-105 plus microsomal fractions, but not in the presence of pure microsomal
fractions (Figure 1). As assessed with Trp-P-2, the activation by either
S-12 or S-105 fractions was interchangeably NADPH- or NADH-dependent, the
mutagenic response being poor when no pyridine nucleotide was included in
the composition of S-9 mix (data not shown).
Figure 1. Metabolic
activation of Trp-P-2 (20 ng/plate), 2-amino-3-methylimidazo[4,5,-f]quinoline
(IQ) (8 ng/plate) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ)
(2 ng/plate) to mutagenic metabolites in strain TA98 of S. typhimurium,
in the presence of varying amounts of liver post-mitochondrial (S-12), cytosolic
(S-105), microsomal, or reconstituted cytosolic plus microsomal fractions
from Aroclor-treated rats. The assays were carried out either in the presence
or in the absence of 50 µM dicoumarol.
As shown in the experiment reported in Figure 2, the cytosolic activation
of Trp-P-2, IQ, and MeIQ to mutagenic metabolites was poor when liver preparations
from either untreated or phenobarbital-treated rats were used; whereas,
metabolism was stimulated by pretreatment of rats with 3-methylcholanthrene
and even more with Aroclor 1254. Again, addition of dicoumarol inhibited
the cytosolic activation of these heterocyclic amines, to a marked extent
in the case of Trp-P-2. Inhibition by dicoumarol was dose-dependent (Figure
3).
Figure 2. Metabolic
activation of Trp-P-2 (20 ng/plate), 2-amino-3-methylimidazo[4,5,-f]quinoline(IQ)
(8 ng/plate) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ)
(2 ng/plate) to mutagenic metabolites in strain TA98 of S. typhimurium,
in the presence of varying amounts of liver cytosolic fractions from either
uninduced, phenobarbital-, 3-methylcholanthrene- or Aroclor 1254-induced
rats. The assays were carried out either in the presence or in the absence
of 50 µM dicoumarol.
Figure 3. Decrease
of Trp-P-2 (20 ng/plate) mutagenicity in strain TA98 of S. typhimurium,
in the presence of liver cytosolic fractions (50 µl/plate) from Aroclor-treated
rats, as related to the amounts of dicoumarol (5-50 µM).
The mutagenicity of Trp-P-2 in the presence of rat liver S-12 or S-105
fractions was similar in the S. typhimurium strains TA98 and TA98NR;
whereas, it was decreased but not abolished (about half in the experiment
shown in Figure 4) in TA98/1,8-DNP6. Inhibition by dicoumarol
was evident and significant (p<0.001, as assessed by Student's
t-test) in all three strains (Figure 4).
Figure 4. Mutagenicity
of Trp-P-2 (50 ng/plate) in strains TA98, TA98NR, and TA98-1,8-DNP6
of S. typhimurium following activation by either S-12 (2.5 µl/plate)
or S-105 (12.5 µl/plate) fractions from Aroclor-treated rats, either
in the presence (dashed columns) or in the absence (open columns) of 50
µM dicoumarol.
Four separate experiments with IQ and one experiment with Trp-P-2 indicated
that addition of purified DT diaphorase, in the presence of either NADH
or NADPH as electron donor, resulted in a poor (from 1.9- to 4.6-fold) yet
reproducible and dose-dependent enhancement of mutagenicity, which was inhibited
by dicoumarol (Table 1).
Similar indications were provided by experiments with the aromatic amines
2AF, 2AAF, and 2AA, shown in Figure 5. All three compounds were activated
not only by S-12 but also by rat liver S-105 fractions, and dicoumarol produced
a significant decrease of mutagenicity.
Figure 5. Mutagenicity
of varying amounts of the aromatic amines 2-aminofluorene, 2-acetylaminofluorene,
and 2-aminoanthracene in strain TA98 of S. typhimurium after activation
by either S-12 (s,d) or S-105 (n,m) fractions from Aroclor-treated rats,
either in the absence (s,n) or presence (d,m) of 50 µM dicoumarol.
Based on preliminary dose-response curves, 10 µl S-12 and 20 µl
S-105 were used for 2AF and 2AA activation, whereas 40 µl S-12 and
80 µl S-105 were used for 2AAF activation.
The activation of Trp-P-2 to mutagenic metabolites was also obtained
in the presence of woodchuck liver S-105 fractions (Figure 6). The results
of these experiments are expressed as relative metabolic efficiency (RME),
i.e., the ratio of mean revertants induced by Trp-P-2 in the presence of
each liver preparation to mean revertants induced by Trp-P-2 in the presence
of S-105 buffer. In spite of a considerable interindividual variability,
activation by liver S-105 fractions from WHV-infected animals (mean ±
SD = 6.8 ± 3.83) was significantly greater than that from uninfected
animals (2.7 ± 0.55) (p<0.05, as assessed by Student's
t-test). The former value did not significantly differ from that
produced by the nontumorous tissue preparations of WHV carriers bearing
PHC (4.6 ± 1.57). Within this group of animals, the liver cytosol
from the cancer tissue was significantly less efficient in activating Trp-P-2
than the surrounding nontumorous tissue (p<0.05, as assessed by
Student's t-test for paired data). Activation by woodchuck liver
cytosol was almost exclusively NADPH dependent, but was not affected by
addition of dicoumarol (data not shown).There was a high (r = 0.88)
and significant (p<0.001) correlation between activation by woodchuck
liver S-105 fractions, as reported in the present study, and activation
by the corresponding S-12 fractions, as it had been reported in a previous
study (13) (Figure 7).
Figure 6. Metabolic
activation of Trp-P-2 (1 µg/plate) in strain TA98 of S. typhimurium
by liver S-105 fractions (amounts corresponding to 2 mg S-105 protein/plate)
from captive woodchuck either uninfected with hepatitis B virus (WHV-) or
infected with hepatitis B virus (WHV+) or additionally bearing primary hepatocellular
carcinoma (PHC+).
Figure 7. Correlation
between metabolic activation by woodchuck liver S-105 fractions (Figure
6) and metabolic activation by the corresponding S-12 fractions (13).
Discussion
The results of the present study provide evidence that liver cytosol
can contribute to the activation not only of aromatic amines, thus confirming
the findings reported by other laboratories (9-11), but also of heterocyclic
amines. The production of mutagenic metabolites by microsomal and cytosolic
fractions was of the same order of magnitude for the two imidazoquinolines;
whereas, cytosolic fractions were even more effective than microsomal fractions
in activating the tryptophan pyrolysis product.
In any case, as assessed with all three heterocyclic amines, the mutagenic
response obtained in the presence of cytosolic and microsomal fractions
was less than additive, as compared to the effect of the original S-12 fractions.
At least in the case of Trp-P-2, activation was even better by reconstituting
cytosolic and microsomal fractions. These results suggest that, besides
an exclusive cytosolic metabolism, an interaction between microsomal and
cytosolic pathways can also occur, such interation being better expressed
when the two subfractions are reconstituted in the experimental system used.
The potentiating effect of the cytosol towards microsomal activation had
been previously reported with aromatic and heterocyclic amines, such as
3-amino-5H-pyrido[4,3-b]indole (18), 2AF (19), and
IQ (20). However, in the last two studies the cytosol alone failed
to activate 2AF and IQ. In another laboratory, where 2AF, 2AAF, and 2AA
had been reported to be activated by the cytosol alone (9), the cytosol
was also found to enhance the S-9 activation of the same compounds (21).
The cytosolic activation of IQ, MeIQ, Trp-P-2, 2AF, 2AAF, and 2AA, as
assessed in the present study, was a) NADPH dependent, b)
uninduced by phenobarbital but induced by 3-methylcholanthrene and, even
more, by Aroclor 1254, and c) inhibited by dicoumarol, with top efficiency
in the case of Trp-P-2. Dicoumarol decreased the mutagenic response induced
in the presence of either S-12 or S-105 fractions but not in the presence
of microsomes. In preliminary assays carried out in our laboratory, the
S-12-mediated mutagenicity of MeIQ and Trp-P-2 and, in addition, of a cigarette
smoke condensate [which among other compounds contains heterocyclic amines
(22) and various quinones (23)] had been found to be decreased
by dicoumarol (24). Inhibition by dicoumarol of S-12 and S-105 activation
of Trp-P-2 occurred in all tester strains, irrespective of their sensitivity
to this compound, which was similar in TA98 and its nitroreductase-deficient
derivative TA98NR, and lower (yet still appreciable) in the O-acetyltransferase-deficient
derivative TA98/1,8-DNP6. This suggests that the results obtained
in the presence of liver subfractions are not affected by further metabolism
in bacterial cells.
All these patterns converge in suggesting a possible role of DT diaphorase
in the cytosolic activation of aromatic and heterocyclic amines. In fact,
this FAD-containing flavoprotein is mostly localized in the cell cytosol,
where it utilizes both reduced pyridine nucleotides as electron donors,
dicoumarol being the most potent inhibitor (12). Moreover, DT diaphorase
is induced in rat liver cytosol by 3-methylcholanthrene and even more by
Aroclor 1254, but not by phenobarbital (25,26 ). However,
an involvement of DT diaphorase in the metabolism of amino compounds is
difficult to be interpreted, because it is obvious that these molecules
cannot per se accept electrons. We raise the hypothesis that the amino group
may undergo a preliminary oxidation to form an intermediate, acting as a
substrate for DT diaphorase. For instance, as shown with pyrolysis products
of tryptophan and glutamic acid, heterocyclic amines can be oxidized in
the cytosol by hydrogen peroxide plus various peroxidases or catalase (27,28).
Alternatively, as reported in the Introduction, amines can undergo a one-electron
oxidation catalyzed by microsomal PHS (7,8), which can explain
a combined microsomal-cytosolic activation of these compounds. It has been
reported that PHS can oxidize 2AF to 2-nitrofluorene (29), and possibly
IQ to nitro-IQ (30).
It is likely that N-hydroxy compounds are common metabolites to
heterocyclic amines and their nitroderivatives, via monooxygenases and nitroreductases,
respectively (31). In this study, however, bacterial nitroreductases
did not affect the cytosolic activation of Trp-P-2. Therefore, we propose
that, following a preliminary oxidation to the nitroso- or the nitro-derivative,
either in the cytosol or in the endoplasmic reticulum, DT diaphorase may
catalyze one or two consecutive two-electron reductions, as it is typical
for this enzyme activity (at least with quinones) (32). The weak
yet consistent generation of mutagenic derivatives following addition of
purified DT diaphorase to IQ or Trp-P-2, in the presence of either NADH
or NADPH, may possibly be ascribed to traces of oxidized amines in the reaction
mixture. It is noteworthy that DT diaphorase has already been shown to metabolize
nitrocompounds, such as 4-nitroquinoline 1-oxide (24,33) and
dinitropyrenes (DNP) (34). In the case of DNP isomers, activation
by liver cytosol contrasts with detoxification by microsomal or S-12 fractions
(34,35). Extensive studies now in progress in our laboratory
show that the mutagenicity of 1,3-DNP, 1,6-DNP, and 1,8-DNP is inhibited
by rat liver S-12 or microsomal fractions, irrespective of Aroclor induction,
as well as by cytosolic fractions from Aroclor-treated rats. The mutagenicity
of 1,3-DNP is considerably enhanced only by using the cytosol from uninduced
rats, with an NADPH-dependent and dicoumarol-inhibitable mechanism. The
mutagenicity of all these compounds is also enhanced by reduced glutathione
(unpublished data).
Trp-P-2 was also activated by the liver cytosol of woodchucks, and cytosolic
activation correlated with S-12 activation, which had been investigated
in a previous study using the same liver specimens (13). The cytosolic
activation was NADPH dependent but was not inhibited by dicoumarol, which
rules out any involvement of DT diaphorase in this rodent species. Therefore,
different mechanisms appear to be involved in the cytosolic activation of
Trp-P-2 in rat and woodchuck liver.
Previous studies using liver postmitochondrial fractions from patients
affected by chronic active hepatitis (36 ), wild-caught woodchucks
affected by chronic active hepatitis (37), and the specimens of captive
woodchucks analyzed in this study (13), had demonstrated that infection
with the specific hepadnaviruses, i.e., hepatitis B virus (HBV) in humans
and WHV in woodchucks, results in an enhanced activation of Trp-P-2 to mutagenic
metabolites. This inducing effect has been confirmed now by using woodchuck
liver cytosolic fractions. Moreover, in agreement with the data obtained
by testing the corresponding S-12 fractions, stimulation of the cytosolic
activation of Trp-P-2 in WHV-infected animals persisted also after PHC formation.
The cancer tissue itself, in accordance with the resistant hepatocyte model
(38), exhibited a reduced Trp-P-2-activating ability, as compared
to the surrounding noncarcinogenic tissue. Since all these effects were
not affected by dicoumarol, and DT diaphorase activity was not altered by
WHV infection (13), other cytosolic mechanisms, such as the marked
depletion of hepatocellular glutathione produced by WHV (13), are
likely to account for the modulation of the cytosolic metabolism of procarcinogens
in viral hepatitis B.
Heterocyclic amines produce higher levels of DNA adducts in the liver
than in other organs, which correlates with their hepatocarcinogenicity
(39). Taking into account that humans are exposed ubiquitously to
these compounds through the ingestion of cooked foods, an enhancement of
their metabolic activation in the liver of hepadnavirus carriers, both in
the cytosol and the endoplasmic reticulum, may bear relevance in the etiopathogenesis
of the PHC forms associated with HBV infection.
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