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:


Dime thy Initr'osamine


Methylethylnitrosamine and higher nitrosamines

Mechanism of carcinogenic action



Life Sciences,  Vol. 27, pp.  2149-2165                        Pergamon Press
Printed in the  U.S.A.


                 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

       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.


                Nitrosamine Bioactivation
Vol. 27, No. 23, 1980

      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

LJ r* u r*
1 ' tin

Vi rj n a-hydroxylation 3N • -
/N N ° NADPH,02 N N 0
H3C • i HO.H2C


i ;• . : n :;/ '
fu ^el ••••- -N2 [;, /. § J -HO®
, L^c J ' • r^c ^-NJ * -.- • •
3ZE \. 3

H C '
N— n


                              Mocromolecular binding
                               FIG'.r I           •
 Activating:metabolic  pathway'of DMN according to the  d^-hydro-
 xylation hypothesis.              "             •

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

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
 .                                                                                                    »
 p-NF         52.3          104.3 +_ 5.9    60.5      221.5  +_ 10.9    53.7        74.4                 H
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**

* Compiled from Refs. 45, 55, 56
**Corrected value based on the difference between  control  tumor  incidences in
  Refs. 45 and 56

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-

Vol. 27,  No. 23, ,1980            Nitrosamine Bioactivation            2155
 raicrosomal  supernatant some DMN-demethylase activity as deter-
 mined  by the  deuterium isotope effect.  The 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

                       :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-

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

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.

      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

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).

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

      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.

2160             Nitrosamine Bioactivation            Vol. 27, No.  23, 1980
     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.

Vol. 27, No. 23, 1980
Nitrosamine Bioactivation

Miciosomul « 	 muiagenesis
^- dealkylases \ carcinogenes
=C2 activation
nature: unknown)
("pH5 enzyme"

\ /
^ Cytoplosmic


• 0
/ ^ \ 	 _
/ * 	 *
In vivo ^
binding ^
nuclear membrane
o X
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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