MINIREVIEW; DIALKYLNITROSAMINE
BIOACTIVATION AND CARCINOGENESIS
David Y. Lai and
Joseph C. Arcos
Prepared for the Chemical Hazard
Identification Branch "Current
Awareness" Program
Invited review, Appeared in
Life Sciences Vol 27, 2149-2165 (1980)
November 1980
-------
-Table of Contents:
Introduction
Dime thy Initr'osamine
Diethylnitrosamine
Methylethylnitrosamine and higher nitrosamines
Mechanism of carcinogenic action
Conclusions
References
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Life Sciences, Vol. 27, pp. 2149-2165 Pergamon Press
Printed in the U.S.A.
MINIREVIEW
DIALKYLNITROSAMINE BIOACTIVATION AND CARCINOGENESIS *
David Y. Lai2 and Joseph C. Arcos
Environmental Protection Agency, Office of Toxic Substances (TS-
792), 401 M St. S.W., Washington, D.C. 20460; and Department of
Medicine, Tulane University Medical Ctr., New Orleans, LA. 70112
Since the initial discovery of Magee and Barnes (1,2,3) that
dimethylnitrosamine (DMN) which had originally been used as
an industrial solvent is hepatotoxic and carcinogenic to a
variety of animal species, the metabolism and carcinogenic action
of nitrosamines have been extensively investigated. In addition
to DMN, several higher nitrosamines and their precursors are
present in the environment. Nitrosamines have been detected in
processed meats (4,5), tobacco and its smoke (6), agricultural
chemicals and cosmetics (7), in urban air (8), and in drinking
water (9).. Thus, they represent an important class of chemical
carcinogens and mutagens potentially hazardous to human health
(10). The aim of this minireview is to present a synopsis of
studies on the mechanisms of metabolic activation and carcino-
genesis of dialkylnitrosamines. A better understanding of the
mechanisms may be helpful in assessing the potential environmen-
tal risk that these chemical agents represent. For more exten-
sive details on the biological effects and environmental hazards
of nitrosamines, the reader is referred to the following articles
(11-14).
The views expressed in this article are those of the
authors and do not necessarily represent those of the U.S.
Environmental Protection Agency or of JRB Associates.
2Under EPA Contract 68-01-4839 with JRB Associates, a
Subsidiary of Science Applications Inc., McLean, Va.
0024-3205/80/492149-17$02.00/0
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2150
Nitrosamine Bioactivation
Vol. 27, No. 23, 1980
Dimethylnitrosamine
The simplest and most common dialkylnitrosamine is DMN; its
metabolism was first studied by Magee (2) in rats, mice and
rabbits. Rapid fall in total body content of DMN after injection
led to the conclusion that its metabolism is fast. Using 14C-
labeled DMN, Dutton and Heath (15) demonstrated that the major
radioactive metabolic product is 14C°;> and concluded that deme-
thylation of DMN takes place and the removed methyl group(s) is
then further oxidized. They also suggested that the biological
effects of DMN may be due to a'metabolite rather than the
compound itself. Studies on the rate of decline of total body
content of DMN in hepatectomized rats, and in rats with induced
hepatic dysfunction, indicated that the liver is the. major organ
that metabolizes DMN (14). . .
• " H3C
N-N=0
AcCX^c'
LJ r* u r*
fee]
|H2C-NsNJ
1 ' tin
iznT
~
Vi rj n a-hydroxylation 3N • -
/N N ° NADPH,02 N N 0
H3C • i HO.H2C
- -HCHO
i ;• . : n :;/ '
fu ^el ••••- -N2 [;, /. § J -HO®
, L^c J ' • r^c ^-NJ * -.- • •
3ZE \. 3
-Hip
H C '
3/N
N— n
H
:M
H3C-N=N-OH
I W
Mocromolecular binding
FIG'.r I •
Activating:metabolic pathway'of DMN according to the d^-hydro-
xylation hypothesis. " •
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Vol. 27, No. 23, 1980 Nitrosamine Bioactivation 2151
In vitro studies of DMN metabolism by Magee and Vandekar
(16), using liver subcellular fractions and tissue slices showed
that the metabolic activity requires C>2 and is localized entirely
in the microsomes plus cytosol fraction. For optimal metabolism
NADPH is required (17,18). These observations were confirmed by
Brouwers and Emmelot (19) who established that formaldehyde is
the main product of in vitro metabolism. Extensive research in
various laboratories led to the currently accepted o(-hydroxyla-
tion hypothesis as the mechanism of metabolic activation of DMN.
According to this hypothesis (FIG. 1) hydroxylation at
the o(-carbon is the critical, rate-limiting first step. The
putative C^-hydroxylated DMN (II) is extremely unstable and
yields, upon hydrolysis, formaldehyde and monomethylnitrosamine
(III); the overall reaction is N-demethylation. Monomethylnitro-
samine (III) is also highly unstable and readily undergoes a
nonenzymatic spontaneous rearrangement or "breakdown" to a
methylating intermediate, which is presumably responsible for the
biological actions of DMN. The evidence that a methylating
intermediate has been formed is the presence, in the carcinogen-
esis target tissues, of proteins and nucleic acids that were
methylated by incorporation of one of the methyl groups derived
from DMN (20-25). However, the chemical nature of the methylat-
ing intermediate is not clearly understood. It has been
suggested at various times that it may represent monomethylnitro-
samine (III) itself (26,27), its tautomeric form, methyldiazonium
hydroxide (IV) (27,28), diazomethane (VII) (29,30) or methyl-
carbonium ion (VI) (21,31,32). Studies by Lijinsky et^ a±. (33)
using deuterated DMN have shown that alkylation of DNA and RNA
involves methylcarbonium ion (VI), without the involvement of
diazomethane (VII). The o(-hydroxylation hypothesis is
supported by the findings that oC-acetoxydimethylnitrosamine
(VIII) (which gives rise to C-hydroxylated DMN upon hydrolysis)
is more carcinogenic, mutagenic and toxic than the parent
compound itself (34,35).
The existence of the enzyme system responsible for the
oxidative demethylation of DMN was reported first by Brouwers and
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2152 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
Emmelot (19). The properties of DMN-demethylase have since been
extensively studied. It is, in several respects, a typical cyto-
chrome P-450-dependent microsomal mixed-function oxidase in that
it requires NADPH and 02 (16,19,36), and can be markedly inhibi-
ted by carbon monoxide (37,38) and repressed by pretreatment with
cobaltous chloride, an inhibitor of the synthesis of cytochrome
P-450 (38). Recent investigations by Lake et_ ja_l_. (39), Arcos et
a.l. (40) and Sipes et al. (41) have revealed that at least two
DMN-demethylase enzyme systems are responsible for the demethyla-
tion of DMN in the liver. One is a low Kj^ enzyme (DMN-demethy-
lase I) and the other a high KJJ, enzyme (DMN-demethylase II). DMN-
demethylase I is regarded to be the enzymic form actually respon-
sible for the in vivo metabolic activation of DMN (40); the
properties of DMN-demethylase I have been extensively investi-
gated and reviewed (38,40-44).
Consistent with the concept that demethylation is the rate-
limiting step for the production from DMN of the toxic "methylat-
ing intermediate", several agents that bring about a lowering of
DMN-demethylase I activity also decrease the carcinogenic activ-
ity and toxicity of DMN (36,45-51). However, recent reports in-
dicated discrepancies in the correlation. For example, Friedman
and Sanders (52) showed that piperonyl butoxide significantly in-
hibits the demethylation of DMN, but does not affect its acute
toxicity or covalent binding to nucleic acids. Similarly, nitro-
sosarcosine, DEN or dibutylnitrosamine decrease the demethylation
of DMN, but without affecting its LD50 in the rat (53,54). We
have shown that in vivo pretreatment with |3-naphthoflavone (3-NF)
pregnenolone -16 0(-carbonitrile (PCN) and 3-methylcholanthrene
(3-MC) decrease the microsome-catalyzed in vitro binding of DMN
to DNA, and this is consistent with their repression of DMN-
demethylase in the rat liver (43,55). However, the p-NF effect
is not consistent with the observation that this compound strong-
ly potentiates DMN hepatocarcinogenesis (TABLE I). In the mouse,
[i -NF increases DMN-demethylase activity, cytochrome P-450 level
and DMN mutagenicity, without affecting the LD5Q (55). PCN in-
creases the P-450 level but decreases DMN-demethylase activity
and DMN toxicity, and has no effect on mutagenicity (55).
-------
TABLE I
Effect of Treatment of Rats with Modifiers on DMM-demethylase
Activity, in vitro Binding and on DMN-induced Hepatocarcinogenicity*
Effect of modi- §
fiers'on DMN -
In Vitro Binding of DMH to DNA induced hepato-
DMN-demethylase Hicrosomes Microsomes + cytospl carcinogenicity
activity (% tumor inci-
Treatment (% of control) pmoles/mg DNA % control pmoles/mg DNA % control dence)
Control 100 172.3 + 6.5 100 412.5 + 12.3 100 32.6 %
— — n
o
Cfi
. »
p-NF 52.3 104.3 +_ 5.9 60.5 221.5 +_ 10.9 53.7 74.4 H
ID
FCIN jy./ luy.J -t- 5.1 bJ.I 1Mb. 5 + 1U.1 4b.^ Ib.b
— ^_
3-MC 54.0 79.8 + 2.0 46.3 128.5 + 9.2 31.1 13.8**
o
tu
o
rt
H-
^
rt
H-
0
* Compiled from Refs. 45, 55, 56
**Corrected value based on the difference between control tumor incidences in
Refs. 45 and 56
Ul
u>
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2154 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
These discrepancies may be partially explained by the
existence of the two enzymic forms of DMN-demethylase. Of these
DMN-demethylase I is repressed and DMN-demethylase II is induced
by pretreatment of the animals with enzyme inducers (40). There
are also speculations that the cytochrome P-450-dependent micro-
somal mixed-function oxidase system might not be exclusively
responsible for the metabolism of DMN, and that possibly
alternative mechanism(s) for the metabolic activation of this
nitrosamine exist. Lake _et_ a^- (39,57-59) suggested that DMN may
be metabolized in part by N-oxidation involving an amine oxidase,
unrelated to cytochrome P-450-dependent mixed-function
oxidases. Their conclusion was based on the findings that: (i)
the storage stability of DMN-demethylase is different from that
of ethylmorphine N-demethylase or 4-chloro-N-methylaniline N-
demethylase, (ii) interaction with DMN produces unique cytochrome
P-450 binding spectra, different from those produced by typical
substrates of mixed-function oxidases, (iii) the demethylation of
DMN is inhibited by benzylamine, a typical substrate of monoamine
oxidase. Argus et al. (56) suggested the possible presence of a
DMN-activating enzyme system in the nuclear membrane with a
differential response to certain inducers, as compared to the
microsomal enzyme. Olah et al. (60) proposed the possible
existence of a protolytic cleavage mechanism in biological
systems leading to the generation of reactive amino-alkylating
intermediate(s). Extensive studies by Lai et al. (55), however,
failed to obtain any experimental evidence for the metabolism of
DMN by either microsomal mixed-function amine oxidase or mono-
amine oxidase. They were also unable to detect any DMN-deme-
thylase or diethylnitrosamine-deethylase activity in the nuclei
or nuclear membrane preparations (55).
Lake ^t_ a^. (58,59) showed that addition of cytosol to the
microsomal preparation markedly increases DMN demethylase
activity and suggested that the cytosol contains substance(s)
that may act as activator(s) of DMN-demethylase, similar to the
effect of cytosol on other microsomal N-demethylases (57,61-
63). Recently, Kroeger-Koepke and Michejda (64) reported the
presence in the so-called "pH 5 enzyme" fraction of the post-
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Vol. 27, No. 23, ,1980 Nitrosamine Bioactivation 2155
raicrosomal supernatant some DMN-demethylase activity as deter-
mined by the deuterium isotope effect. The enzyme.in the
postmicrosornal supernatant was, shown to be different from that in
the rnicrosomal pellet and has different characteristics in two
different strains of rats. The increase in the binding of DMN to
DNA by the addition of cytosol, observed by Chin and Bosman (65)
and by Lai _e_t_ al_* (55), appears to be consistent with these find-
ings. However, the effect of mixed-function .oxidase modifiers on
this soluble enzyme and its metabolic relationship to the two
endoplasmic reticulum-localized DMN-demethylases remain to be
investigated.
:Diethylnitrosamine •
In contrast to the considerable number of studies on DMN,
there has been until recently lesser interest in the metabolic
activation of diethylnitrosamine (DEN) and other dialkylnitrosa-
mines. For DEN,;monodeethylation followed by the production of a
reactive ethonium ion from the remainder of the molecule, monoe-
thylnitrosamine, appears to be the pathway of bioactivation. The
properties of DEN-deethylase have been studied- by several inves-
tigators (17, 18, 66-68). Magour and Nievel (66) have shown that
deethylase activity is enhanced by pretreatment of animals with
3-MC, phenobarbital (PB), butylhydroxytoluene and DDT [1,1,1-tri-
chloro-2,2-bis(j>rchlorophenyl) ethane] . Arcos^eJ^ al_. (67) confirm-
ed the inducing effect.of PB, but found 3-MC pretreatment to have
an inhibitory effect, and they suggested the possible existence
of more than one form of DEN-deethylase responding differently to
3-MC treatment. Chau,j2t_aK (68) reported that the Km for DEN-
deethylation is an order of magnitude smaller than that of DMN.
Diethylnitrosamine metabolism does not lead to the formation of
any formaldehyde, suggesting that in vitro metabolism of DEN
occurs exclusively by iX-oxidation, yielding acetaldehyde (67).
The relevance of deethylase activity to hepatocarcinogenesis
has been studied by Rao and Vesselinovitch (69) who described a
positive correlation between the degree of DEN-deethylation and
susceptibility to hepatocarcinogenesis in mice, as a function of
age. Montesano and Magee" (70,71) have studied the organ distri-
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2156 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
bution of DEN-deethylase activity in the rat and the hamster, and
observed a positive correlation between deethylase activity and
the relative susceptibility of the various organs to DEN carcino-
genesis. The finding that PB significantly increases, whereas 3-
MC decreases the microsome-catalyzed binding of DEN to DNA (61)
are consistent with these drug effects on the metabolism of DEN
(67). The observation (61) that addition of cytosol enhances the
binding of DEN suggests that activating substance(s) of microso-
mal DEN-deethylase may be present in the cytosol.
The possibility that DEN may be ^3-oxidized and dehydrated to
vinylethylnitrosamine or oxidized to an unknown bifunctional
derivative, which would interact with cellular macromolecules by
cross-linking, has been hypothesized (72,73); however, there is
as yet no experimental evidence for this. The observation (61)
that addition of mitochondria stimulates the microsome-mediated
binding of DEN and N-nitrosopiperidine and, furthermore, that
mitochondria alone can catalyze the binding of N-nitrosopiperi-
dine support the possibility that nitrosamines may be metabo-
lized, at least in part, by enzymes unrelated to cytochrome P-
450-dependent mixed-function oxidases. The mitochondrial
enhancement of binding appears to be a function of the alkyl
chain length of the nitrosamine, since the mitochondrial stimu-
lation of binding is nil with DMN, small (1.4-fold) with DEN, but
considerable (4-5 fold) with N-nitrosopiperidine (61). The bind-
ing of N-nitrosopiperidine was further shown to be strongly re-
duced by benzylamine, a typical substrate of mitochondrial monoa-
mine oxidase (61). This suggests that amine oxidase pathway(s)
probably play a role in the metabolism of DEN and other higher
nitrosamines, but DMN does not appear to be a substrate for
either microsomal or mitochondrial amine oxidase (55,74).
Methylethylnitrosamine and Higher Nitrosamines.
Chau et^ &!_. (68) have studied the kinetics of oxidative
dealkylation of methylethylnitrosamine (MEN) by rat liver
microsomes using a colorimetric assay for the simultaneous
analysis of formaldehyde and acetaldehyde. Both MEN-demethylase
and -deethylase are stimulated by PB (68,75) and inhibited by
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Vol. 27, No. 23, 1980 . Nitrosamine Bioactivation
3-MC (75) pretreatment. The Km for MEN-demethylase and -deethyl-
ase are 48mM and 75mM, respectively (68).
Upon testing the carcinogenicity of a series of MEN that are
deuterated at 'different sites, Lijinsky and Reuber (76) showed
that the carcinogenicity of these compounds increases consider-
ably with the increasing deuteration of the ethyl group. Since
the C-D bond is much stronger than the C-H bond, they concluded
that oxidation of the ethyl group at either carbon is unrelated
to tumor induction by MEN.
More recently, Lai ^t_ al^. (75) showed that rat liver
microsomes catalyze substantially the covalent binding of C-MEN
to DNA. Converging lines of evidence in the study suggest that
the reactive intermediate which binds to DNA is ethylcarbonium
ion derived from monoethylnitrosamine. This is consistent with
the view of Lijinsky and Reuber (76) that ethylation rather than
methylation is the critical molecular event in liver carcino-
genesis by MEN.
The biotransformation and metabolic fate of dialkylnitrosa-
mines higher than DEN is considerably more complex and is being
extensively studied presently. Studies by Kruger (77) suggest
that higher dialkylnitrosamines are metabolically degraded by /3-
-oxidation (analogous to fatty acid metabolisms) to methyl-alkyl
or dimethylnitrosamine, which then react as the methylating
agent. Investigations by other workers indicate that hydroxy-
lation of higher dialkylnitrosamines at carbon atoms other than
the c( and ft positions also occurs. The reader is referred to the
papers of Okada (78) and Blattmann and Preussmann (79,80) for
details on this study area.
MECHANISM OF CARCINOGENIC ACTION
Covalent binding of reactive intermediates of carcinogens to
cellular macromolecules is generally considered to be the mecha- .
nism initiating carcinogenesis by most chemical agents. The
consensus is that infidelity of DNA replication as a result of
modification of DNA by the chemical agents is the basis of
carcinogenesis.
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2158 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
In vivo covalent binding to DNA of a methyl group of DMN was
first demonstrated by Magee and Farber (23). Since then a large
number of reports have appeared showing covalent binding of
various alkylcarbonium ions originating from dialkylnitrosamines,
under both in vivo and in vitro conditions (24,61,65,81-34). The
predominance of methylation of guanine at the N7-position has led
to the suggestion early that this reaction may have potential
significance in carcinogenesis. However, the degree of alkyla-
tion at the N -position of guanine showed no correlation with the
carcinogenic activities (85-87). Moreover, in a study of the
properties of 7-methylguanine-containing templates, Ludlum (88)
showed that the methylated base can still pair normally with
cytosine. Moreover, the coding properties of DNA-containing
guanine residues substituted in the 7-position were not lost,
indicating that alkylation of the nucleic acid at this site may
be of little biological significance.
There is increasing evidence that alkylation at the 0 -
position of guanine may be responsible for the carcinogenic
activity of nitrosamines (25,89-91). The formation and persis-
tence of 0 -alkyl derivatives in DNA after treatment with N-
nitroso carcinogens have recently been reviewed by Pegg and
Nicoll (92) and by Singer (93). An important piece of evidence
for the biological importance of 0 -alkylation as opposed to N •-
alkylation was provided by Loveless (94): deoxyguanine was
methylated in vitro at the 0 -position by nitrosourea, but not by
, j
the non-carcinogenic methylmethanesulfonate. Similarly, evidence
for the formation of 0 -methylguanine was found in cells treated
with N-methyl N'-nitro N-nitrosoguanidine, but not in cells
treated with dimethylsulfate, which is relatively non-carcinogen-
ic under these conditions (95). A positive correlation between
the formation of 0 -alkylguanine and the ability to induce muta-
tion in phage has also been observed (94,96,97). In the studies
of the properties of 0 -methylguanine-containing template,
Gerchman and Ludlum (98) showed the incorporation of "incorrect
nucleosides" by bacterial RNA polymerase. It was suggested that
06-alkylation leads to the inability of the guanosine residue to
undergo normal base-pairing with cytosine and, thus, may lead to
transition mispairing resulting in mutation (94,98).
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Vol. 27, No. 23, 1980 • Nitrosamine Bioactivation 2159
Studies by various investigators have recently established
that tissues which readily develop tumors after a single dose of
carcinogen, have repair mechanisms which are much less capable of
removing the 0 -alkylguanine from their DNA, than other
tissues. For instance, after a single large dose of DMN, which
produces kidney tumors but not liver tumors, 0 -methylguanine was
found to have a much longer half-life in kidney DNA than in liver
DNA (89-91). Similarly, after treatment of rats with low doses
of ethyl or methylnitrosourea, 0 -alkylguanine was found to be
more persistent in the DNA of the brain, in which tumors are
obtained (99,100). Thus, it appears that the properties of the
repair system capable of removing 0 -alkylguanine from DMA before
cell division is important in determining organ susceptibility to
the carcinogenic stimulus of nitrosamines and other alkylating
carcinogens.
An enzyme that catalyzes the release of 0 -methylguanine as
free base from alkylated DNA has been isolated from J2. coli (101)
as well as from the liver and kidney of the rat (102). Recently,
evidence has been reported that cells which become malignant
after treatment with nitrosamines are deficient in repair enzyme
activity (90,91,103,104). The report by Pegg (105) on an enzyme
that excises 0 -alkylguanine but not N -alkylguanine emphasizes
the biological significance of 0 -alkylation.
Pegg and Hui (102) observed that the removal of 0 -methyl-
guanine from DNA was much more efficient after low doses of DMN
than higher doses. Thus, it was suggested that the repair enzyme
system itself may be the target for inactivation by large doses
of the carcinogen. Although little is known about how the
modification of enzymes and other proteins by alkylating agents
affect transcription and translation, the modification could lead
to: (i) alteration or inactivation of repair enzymes; (ii)
infidelity of DNA replication as a result of the alteration or
inactivation of DNA polymerase; (iii) changes in gene expres-
sion; and (iv) derepression of part or all of integrated tumor
virus genomes or oncogenes.
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2160 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
CONCLUSIONS
It is widely accepted that prior metabolic activation of
nitrosamines by various oxidases to alkylating agents which react
with cellular macromolecules (DNA and/or RNA) is required for
their carcinogenic or mutagenic activity.
In vitro studies (14,16) Have demonstrated that they are
primarily metabolized by cytochrome P-450-dependent microsomal
mixed-function oxidases in the liver. At least two distinct
forms of DMN-demethylase have been shown to demethylate DMN
(40). The possible existence of more than one form of DEN-
deethylase has also been suggested (67).
Despite earlier reports to the contrary, the postmicrosomal
soluble cytosol itself appears to contain DMN-demethylase activ-
ity, present in the "pH 5 enzyme" fraction (64), as well as
activating substance(s) of DEN-deethylase.. Moreover, mitochon-
drial enzyme system(s) appear to be involved in the bioactivation
of nitrosamines higher than DMN (61).
These mixed-function oxidases are subject to induction and
repression by various agents and, thus, they may influence the
bioactivation and carcinogenesis by nitrosamines. Moreover,
these modifiers may influence nitrosamine carcinogenesis by
mechanism(s) entirely unrelated to the enhancement or inhibition
of nitrosamine metabolism. The mechanisms could include: effect
on the rate of cytoplasmlc transport and stabilization of the
reactive intermediate(s), the rendering of specific macromole-
cular sites more or less available to alkylation by acting on DNA
conformation-modifying enzymes, inhibiting or enhancing DNA
repair activity, etc. The evidence so far available suggest
that p-NF may be one of the agents which enhances DMN carcino-
genesis by a mechanism unrelated to its effect on DMN metabolism
(43,56). The presently perceived interrelationship of factors
influencing nitrosamine metabolism and binding to DNA is
illustrated in FIG. 2.
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Vol. 27, No. 23, 1980
Nitrosamine Bioactivation
2161
L
activation
mechanisms
Modifiers
Miciosomul « muiagenesis
^- dealkylases \ carcinogenes
1
1
Mitochondrial
=C2 activation
(enzymatic
nature: unknown)
1
1
Cytoplasmic
DMN-demethylase
("pH5 enzyme"
group)
\ /
^ Cytoplosmic
/stabilization
of
and
is
a
• 0
D
%
/ ^ \ _
/ * *
In vivo ^
binding ^
toONA A
nuclear membrane
i
i
1
o X
n
z
1
1
ONA conformation
modifying enzymes
FIG. 2
The quantitatively predominant site of alkylation of
(and RNA) by nitrosamines is the N -position of guanine. For
this reason, the formation of this product was believed for some
time to be the key molecular event in nitrosamine carcinogenesis.
However, recent experimental data do not support this view. The
accumulated evidence shows that the formation and persistence of
06-alkylguanine may be the key factor for tumor initiation, and
that the differing repair enzyme activities in various organs may
account for the varying susceptibilities of tissues to the
carcinogenic stimulus. However, the interaction of alkylating
agents with proteins cannot as yet be ruled out as playing some
role in the induction of tumors.
Acknowledgements . Background information used in this review was
acquired as an outcome of research, supported by The Council for
Tobacco Research (Grant 922A) , during the period 1974-1980,
carried out at Tulane Medical Center.
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2162 Nitrosamine Bioactivation Vol. 27, No. 23, 1980
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