(CANCER RESEARCH 50. 1144-1150. February 15.
I990|
Metabolic Denitrosation of jY-Nitrosodimethylamine
Anthony J. Streeter, Raymond W. Nims, Pamela R. Sheffels, Young-Hun Heur, Chung S. Yang, Bruce A. Mico,
Charles T. Combar, and Larry K. Keefer'
Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 [A. J. S., R.
W. N., P. R. S., Y-H. H., L. K. K.J; Department of Chemical Biology and Pharmacognosy, Rutgers University, Piscataway, New Jersey 09955 [C. S. Y'J; Department of
Drug Metabolism, Hoffmann-La Roche Inc., Nutley, Nett' Jersey 07110 ¡B.A. M./; and Department of Drug Metabolism, Smith, Kline & French Laboratories,
Philadelphia, Pennsylvania 19101 [C. T. G.j
ABSTRACT
Enzymatic denitrosation is a potentially inactivating metabolic route
that has been shown to convert carcinogenic /V-nitrosodimethylamine
(NDIV1A)to methylamine (MA) in vitro. To investigate its quantitative
course in vivo, groups of 8-week-old male Fischer rats have been given
small (8-15 ¿tmol/kg)p.o. or i.v. bolus doses of uC-labeled NDMA and
the subsequent formation of radioactive MA has been monitored by high
performance liquid Chromatographie analysis of serially collected blood
samples from each individual. Adjusting the |"( |M \ fluxes observed for
the previously measured rates at which MA is itself eliminated from the
system after intragastric administration, denitrosation was calculated to
represent a rather uniform 21.3 ±1.3% (SE) of total NDMA elimination
in the four animals studied. By contrast, repetition of the experiment
with fully deuterated NDMA (NDM V-</,,)revealed a significantly wider
variance in the results (39.8 ±8.9%). An alternative calculation using
values for elimination of i.v. doses of MA and its trideuteromethyl
analogue gave an even larger difference for MA formation between
NDMA and M >MA-</„, the estimated extents of in vivo denitrosation in
this case being 14.5 ±0.9% and 48.3 ±10.8%, respectively. The results
indicate that denitrosation is a major metabolic pathway for NDMA
elimination and suggest that deuteration of the carcinogen induces a shift
in its metabolism toward increasing denitrosation at the expense of the
competing activation pathway. Consequently, denitrosation may be the
previously undefined in vivo metabolic route, the existence of which was
suggested by the findings that deuteration of NDMA lowered its hepa-
tocarcinogenicity and liver DNA alkylating ability in rats.
INTRODUCTION
The potent hepatocarcinogen (I), NDMA,2 is metabolized by
rat liver microsomes via two competing mechanisms (2-6), as
shown in Fig. 1. One is the activating dealkylation route de
picted in path a, which leads to the formation of formaldehyde,
dinitrogen gas, and a potent methylating species. The other is
the transformation referred to as enzymatic denitrosation,
which is summarized in path b and is presumed to inactivate
the carcinogen. The latter pathway results in the production of
formaldehyde, monomethylamine, and nitric oxide which is
oxidized to nitrite under the microsomal incubation conditions.
While the denitrosation mechanism has been studied fairly
extensively in vitro (2-7), only a few tentative conclusions about
its course in the intact animal have appeared thus far in the
literature (8, 9). Formaldehyde is unsuitable as an indicator of
the extent to which either route may be utilized since it is a
Received 7/5/89; revised 11/6/89; accepted 11/14/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, c/o NCI-FCRF. Bldg.
538. Frederick. M D 21701.
1The abbreviations used are: NDMA. ,\'-nitrosodimethylamine; MA. mono
methylamine; NDMA-rf,. ,V-nitrosodi(|:H,]methyI)amine; MA-</,. mono[2Hj|-
methylamine: DMA, dimethylamine; HPLC. high performance liquid chroma-
tography; i.g.. intragastric; «and ¡1.
first order rate constants for the elimination,
distribution, or absorption phases; AUC. area under the blood concentration
versus time curve; CI, systemic blood clearance; C/».renal blood clearance: C7H. zinc sulfate solution followed by 71 ^1 of saturated barium hydroxide
hepatic blood clearance; C'A, intrinsic hepatic clearance; Q, hepatic blood flow
rate; F, systemic bioavailability: M") and MJ). half-lives of the elimination,
distribution, or absorption phases: l'ss. apparent steady state volume of distribu
tion.
Downloaded from cancerres.aacrjournals.org on April 3, 2020. © 1990 American Association for Cancer Research.
in Vivo in the Rat
common metabolite for both pathways and, anyway, it is rapidly
and extensively oxidized to carbon dioxide in the intact animal
(10). Metabolically produced nitrite is completely converted to
nitrate in vivo (11). While it is possible to distinguish the nitrate
derived from nitrosamines in vivo from the endogenous nitrate
arising from other sources by using mass spectral techniques if
the administered compound is "N-labeled (12), such method
ology becomes increasingly difficult as the dose is lowered.
Consequently, the most direct assessment of the denitrosation
pathway should come from measuring the extent of conversion
of NDMA to MA, and if the NDMA is labeled with I4C the
metabolic MA can be easily differentiated from the endogenous
material which is known to be present (13).
This paper reports experiments aimed at confirming the
occurrence of the denitrosation pathway in vivo in the rat and
measuring its extent using more appropriate methodology than
has previously been brought to bear on the problem. In addition,
quantification is presented which describes the effect of com
plete substrate deuteration on the balance between dealkylation
and denitrosation in vivo, which was carried out in an effort to
elucidate the origins of the previously reported deuterium iso
tope effect on the hepatocarcinogenicity of NDMA (14).
MATERIALS AND METHODS
Chemicals. Unlabeled MA hydrochloride and DMA hydrochloride
were purchased from Aldrich Chemical Co. (Milwaukee, WI), NDMA
was bought from Sigma Chemical Co. (St. Louis, MO), and NDMA-
</6was synthesized as published previously (14). [UC]MA hydrochloride
(8.9 mCi/mmol) was obtained from New England Nuclear (Boston,
MA), [UC]DMA hydrochloride (58 mCi/mmol) was from Amersham
(Arlington Heights, IL), and 114C]MA-rf,( 19.8 mCi/mmol) was synthe
sized as described elsewhere (15). [I4C]NDMA (40 mCi/mmol) and
[14C]NDMA-i/6 (40 mCi/mmol) were prepared by SRI International
(Menlo Park, CA) and were shown to have radiochemical purities of
99.8 and 99.2% as determined by HPLC analysis shortly before use.
Sodium 1-heptanesulfonate was purchased from Eastman Kodak Co.
(Rochester, NY).
Animals and Treatments. Male Fischer (F344/NCr) rats were ob
tained from the Animal Production Area of the Frederick Cancer
Research Facility, housed on wire grids, acclimatized to a 12-h light/
dark cycle (7 a.m./7 p.m.) for about 1 week, and given access to food
(NIH autoclavable formula 31 modified to contain 6% fat; Zeigler
Bros., Gardners, PA) and water ad libitum until they were used at 54
±1 (SE) days of age. A single dose of either [I4C)NDMA (40 mCi/
mmol) or ('4C)NDMA-</6 (40 or 8.9 mCi/mmol) was given to each rat
by gavage or i.v. bolus injection into a tail vein (as indicated in Tables
1 and 2) between 8:40 a.m. and 1:44 p.m.
Concomitant Assay of NDMA and MA in Blood. Approximately 20
serial 50-jjl retroorbital blood samples from each rat were collected
into weighed 1.5-ml microcentrifuge tubes containing 20 u\ of heparin
solution (100 units/ml). Each tube was reweighed to determine the
volume of blood added, using a value of 1.05 g/ml as the density of the
blood. The protein was then precipitated by the addition of 70 M'of 5%
solution. The precipitate was sedimented in an Eppendorf model 5414
microcentrifuge for 5 min. Samples of 25 u\ of supernatant were taken
for liquid scintillation counting, while separate aliquots of 70 ^1 of each
1144
 M VIVO DENITROSATION OF /V-NITROSODIMETHYLAMINE

were then assayed by HPLC for both NDMA and MA by quantifying HOCH,
N—NO CH3X » N2 - CH,O * OH
the peaks of radioactive material that eluted at the retention times of CH,'
CH3
authentic NDMA and MA and comparing those values to standard N-NO
curves constructed by spiking blood samples with |'4C]NDMA or [14C]- CH,'
MA. The respective deuterated compounds were used for the standard
curves when NDMA-rf6 was administered to an animal. CH,
N
The isocratic HPLC system used was a slight modification of one II CHjNH, - CH2O
which is described in detail elsewhere (16). Briefly, the system consisted CH2
of a Waters model 510 pump, a Waters model U6K injector, and a
model 1C Flo-one\/3 radioactive flow detector (Radiomatic Instru
[01 10]
•¿NO NO,
ments, Tampa, FL). Four HPLC columns were used in series. They NO_

were, in order, a 5-cm x 4.6-mm guard column (packed with 10-Mm

C|g particles obtained from Supelco Chromatography Supplies, Belle- Fig. 1. Known mechanisms of NDMA metabolism as adapted from Ref. 6.
I'mh a, the dealkylation pathway leading to the ultimately genotoxic methyl
fonte, PA), a 25-cm x 4.6-mm Spherisorb ODS2 column (5 ^m; Anspec
diazonium ion. Path b, the course or enzymatic denitrosation, a potentially
Co., Ann Arbor, MI), a 5-cm x 11-mm column (packed with the cation inactivating elimination route that proceeds in parallel with dealkylation.
exchange resin Aminex A-7, 9 ^m; Bio-Rad Laboratories, Richmond,
CA), and a 10-cm x 11-mm column (packed with the aniónexchange
resin Aminex A-27, 15 ^m; Bio-Rad). The system was eluted with 200
mM ammonium phosphate buffer (pH 3.0) at 1.0 ml/min, and the
A
radioactive flow detector added scintillation cocktail (Flo-Scint II;
Radiomatic Instruments) to the effluent from the final column at 2.0 10.0-
ml/min. The injection of '4C-labeled standards revealed retention times
in this system for the potential metabolites formaldehyde, monometh-
lurea, and formic acid of 7.6, 9.0, and 16.6 min, respectively. The o o
retention times of NDMA and MA were found to be 19.4 and 29.3 o o
min, respectively. Because of the length of the HPLC assays, the
samples from each rat were divided into two groups by alternate blood
1.0-
sampling times, and each group was assayed on a single day with its
own standard curves.
Renal Excretion. Separate groups of rats were used to estimate the
total renal excretion. Each rat was given an i.v. bolus dose of [I4C]-
NDMA (40 mCi/mmol) or [l4C]NDMA-</6 (8.9 mCi/mmol), as indi 0.1
cated in Table 2, and was then placed in an individual polycarbonate O 10 20 30 40 50 60 70
metabolism chamber to allow for the separate collection of urine. The
animals were given access to food and water ad libitum. Urine was
collected over 0-24-h, 24-48-h, and 48-72-h periods after dosing. B
Samples were taken for liquid scintillation counting to determine the
total amount of radioactivity excreted, and aliquots of 100 ¿tlwere
subjected to the assay for NDMA and MA described above. 10.0-
Metabolite Identification. Since it has been ascertained that DMA cr o o
also has the same retention time as MA in the above mentioned HPLC 0)
0 O o
assay, it was necessary to verify that the metabolite quantified was
actually MA, even though DMA has been reported previously not to
be a microsomal metabolite of NDMA in the rat (4, 6). 1.0-
Because the amounts of radioactive metabolite found in blood sam
ples were too small to allow use of the isotope dilution technique, a
•¿â€¢â€¢
separate HPLC method was developed. The equipment was similar to
that described above except that only a single HPLC column was used
(Spherisorb ODS2, 25 cm x 4.6 mm, 5 urn; Anspec Co.) which was
eluted at 1.0 ml/min with 7 mM ammonium phosphate buffer (pH 3) 0.1
containing 5 mM sodium I-heptanesulfonate. 10 20 30 40 50 60 70
Injections of '4C-labeled standards revealed retention times of 10,
17, and 24 min for NDMA. MA, and DMA, respectively. The other Time following i.v. administration (min)
potential metabolites for which standards were available gave peaks Fig. 2. Blood concentrations of unchanged compound (A), monomethylamine
close to the void volume of the system at 4.0, 4.0, 4.4, and 4.6 min for (•),or total radioactivity (O) for typical animals as a function of time after a
single i.v. bolus administration of [I4C]NDMA to a male Fischer 344 rat at adose
formaldehyde, formate, methanol, and monomethylurea, respectively. of 7.6 ^mol/kg (A) or a single i.v. bolus administration of |"C|NDMA-d6 to
Toxicokinetic Analysis and Protein Binding. The parameters were another rat at a dose of 7.0 nmol/kg (B).
obtained as described previously (17).
the tissues. Such a conclusion is also supported by the additional
RESULTS observation that the a phase seen for the total radioactive
material in the blood after i.v. injection of NDMA or NDMA-
The plots of concentration of unchanged compound in whole i/6 overlaps that of the unchanged compound (Fig. 2; Table 1).
blood versus time following a single i.v. bolus dose revealed However, inspection of the subsequent elimination (ß) phases
biphasic toxicokinetics for both NDMA and NDMA-i/6 (Fig. for unchanged compound and total radioactivity following an
2), and for the first time the initial (a) phase of NDMA was i.v. bolus dose of NDMA demonstrated that the unchanged
adequately characterized in the rat using the retroorbital blood compound was being removed from the systemic circulation at
sampling technique (Table 1). No differences were found in the a much greater rate than the total radioactivity, indicating that
initial phase upon deuteration, which is as expected if this phase metabolites were being eliminated more slowly than they were
represents a distribution of the compound from the blood into being formed. We were able to identify unambiguously one of
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 IN VIVO DENITROSATION OF AMMITROSODIMETHYLAMINE

Table 1 Toxicokinetic parameters for NDMA and NDMA-dt, methylamine produced metabolically from them, and total radioactivity obtained by serially sampling the
blood of male Fischer rats following bolus doses of the carcinogen
NDMAParameterBody

administration administration administration administration

4)171
<JV= 4)190
(N = 6)203
(N = 4)176+
(N =
(g)Dose
wt 18°8.36
± 413.5+ 78.31+ 1014.6
(nmol/kg)Unchanged 1.0516.6
± ±0.33.36 0.9516.6
+ 0.36.52
+

nitrosamineA4
(nmol/ml)B ±3.08.31 0.961.77
± 2.813.3
+ 0.46'5.03
±
(nmol/ml)a ±0.540.657 ±0.200.753 2.60.999
+ Q.2S"0.410
±
(min"1)il
±0.1010.0653 ±0.1800.0493 450.0470±0.1 ±0.0370.0298
(min"')M«) 0.0022"0.793
± 0.0021'1.73
+
0.00251.14
± ±0.00511.12
(min)MÃ-i) ±0.1710.7 0.2914.5
+ 0.14414.9
+ ±0.1423.6+
(min)AUC 0.4166
± 1.335.5
+ 0.87309
± 1.7'157
min/ml)CI/F
(nmol 1050.2± 3.6388
± 53'28.4
+ ±8''94.4
(ml/min/kg)Y„ 4.8630
± 2924.1
± ±2.^563 6.3*38.3
±
(ml/kg)MRT 4412.7
± 2720.2
+
(min)MAT 0.70.631
+ +2.311.4 \Ar0.795
± 2Af18.1
±
(min)FMethylamine 2.30.13
+ ±2.40.31
±0.010.346 ±0.02''7.17

metaboliteB
(nmol/ml)/} ±0.0550.0265 0.0540.0109
± 0.1830.00598
+
(min"')MiO ±0.00150''191
0.001626.5
± 0.002074.3
±
(min)AUC 1.822.8
± 19.547.6
± 73«166±40/'*17.8
+
min/ml)Total
(nmol 2.616.4
± 14.36.13
+

equivalents)A
radioactivity (NDMA
(nmol/ml)B 2.78.05 1.213.49
± 2.610.3
+ 0.395.80
(nmol/ml)« 0.650.868 50.797 ±0.1 1.50.924
± 0.310.351
(min"'),i 0.1270.0289 ±0.2170.0123 0.0810.0+ 0.0450.00579
(min"')i»(n) 0.00071.26
+ ±0.0005''0.779
177 0.00062''2.10
0.00100.855
(min)/„(#) 0.13024.1 0.2756.7
+ 0.06739.4+
± 0.31124
(min).v. 0.8¡.g- + 2.7NDMA-rf6i.V. 1.0"'•g- \Sf
' Mean + SE.
* A and B. coefficients of the exponential terms which describe the change in blood concentrations over time; MRT. mean residence time; MAT, mean absorption
time.
' P < 0.05. significantly different from nondeuterated compound (Student's t test).
* P < 0.001, significantly different from nondeuterated compound (Student's I test).
' P< 0.02, significantly different from nondeuterated compound (Student's t test).
1P < 0.01. significantly different from nondeuterated compound (Student's / test).
* P < 0.02, significantly different from nondeuterated compound (Mann-Whitney U test).

the metabolites as MA by its Chromatographie properties on in agreement with the rapid conversion of NDMA to MA, since
the multiple column HPLC system (16) used for the analysis the much more rapid disappearance of NDMA would not
of all of the blood samples, and on an alternative ion-pairing hinder the elimination of MA and is also consistent with the
system, capable of distinguishing between MA and DMA, existence of only short-lived intermediates in the formation of
which was used to check selected samples. It was considered MA such as /V-methylformaldimine (16).
necessary to use the latter system since DMA could conceivably The administration of NDMA-rf6 to rats by i.v. bolus injection
have been formed from NDMA in vivo in the rat. resulted in significantly longer r./,(/3)and mean residence time
Scrutiny of the individual HPLC traces from the later blood and significantly smaller systemic blood clearance for the un
samples revealed that, in addition to NDMA and MA, there changed compound when compared to the undeuterated com
were large amounts of radioactive material which were chro- pound (Table 1; Fig. 2). A similar increase was seen in the tv,(ß)
matographing at a retention volume only slightly greater than for total radioactivity. In the case of metabolic MA, the concen
the void volume of the system. While this material was chro- tration of deuterated MA reached a higher maximum and
matographing at about the same time as would be expected for decreased more slowly than undeuterated MA because of the
formaldehyde, the shapes of the peaks indicated that more than large kinetic isotope effect on the further metabolism of MA
one component may have been present. Consequently, it was (15). Following a single i.g. dose administered by gavage (Fig.
not possible in the present study to quantify any circulating 3; Table 1) curves for blood concentration of NDMA or
metabolite other than MA. NDMA-t/6 versus time were obtained which, when subjected to
A plot of the blood concentration of metabolic MA versus the method of residuals, revealed only two distinguishable ex
time is shown in Fig. 2. In calculating the concentrations of ponential phases. Comparison of the AUC values to those of
MA from the amounts of radioactivity measured at the appro the respective i.v. bolus plots revealed systemic bioavailabilities
priate retention time, it was assumed that the presence of a 14C of 13 and 31% for NDMA and NDMA-d6, respectively. When
atom on one of the methyl groups did not influence which the profiles of total radioactivity versus time were compared to
methyl group was oxidized and, therefore, that the specific those of unchanged compound, there was again a more rapid
activity of the resulting [I4C]MA was exactly one-half that of elimination of the unchanged compound in each case. MA was
the administered [UC]NDMA. detected as a metabolite of both compounds but, while its
The similarity of the terminal half-life for MA when formed concentration was seen to peak early and then decline in the
as a metabolite of NDMA (Table 1) to that obtained when MA case of the undeuterated compound, MA-rf, was observed to
itself was dosed i.v. (15) confirms that the clearance of MA is increase to a plateau with no discernible decrease during the
limited by elimination rate and not by formation rate. This is course of the experiment with NDMA-rf6. The latter phenom-
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 IN VIVO DENITROSATION OF /V-NITROSODIMETHYLAMINE

10.00 as in our earlier report and with similar results (18). The
calculation of isotope effects based on the present data revealed
values of 1.3, 1.8, 2.4, and 4.1 for first-pass metabolism, sys
°°°°° ° o
0 0 temic blood clearance, bioavailability, and intrinsic hepatic
0 0
clearance (CA), respectively. The value for Cl\ can be calculated
as Cl/F after an i.g. dose and is a direct measure of the Vmax/
1.00:
-o Km ratio at the enzyme level, since NDMA is completely
o
o absorbed (20, 21), is metabolized predominantly in the liver
(22), undergoes first order elimination and is protein bound to
a negligible extent. The value of 4.1 is in excellent agreement
o
with the figure of 3.9 obtained for the (Vmall/A:m)H/(Vmax/#m)D
C 0.10 ratio by measuring the initial rates of nitrosamine metabolism
«^^
C
following concurrent administration of NDMA and NDMA-¿6
o 0.05 (23).
10 20 30 40 When NDMA was given by i.v. administration to rats at a
C
O) dose of 1 mg/kg, no unchanged compound was detectable in
o 10.00
C
o
the urine (Table 2). This is not unexpected as detailed studies
o have reported that very low urinary NDMA levels were found
-*->
C
ju at such low doses, which do not saturate the metabolizing
o enzymes (24). MA was found as a urinary metabolite, however,
>
'zi
cr and when appropriate calculations were implemented to correct
0)
1.00- for the different specific activities, it was seen to account for
»
*»«
* »»» the majority of the radioactive material excreted in the urine
(Table 2). MA has been reported previously as a urinary metab
olite of NDMA (9, 25). When NDMA-i/6 was administered to
another group of rats, approximately 0.1% of the dose was
recovered unchanged in the 0-24-h urine collection. This is
0.10- equivalent to a renal blood clearance of 0.03 ml/min/kg, but
such a small amount has a negligible effect on the overall
0.05 systemic blood clearance. Deuteration also resulted in signifi
10 20 30 40
cantly greater renal excretion of total radioactivity and mono-
Time following ¡.g. administration (min) methylamine, with MA again accounting for the largest part of
the radioactive material. The urinary metabolic MA data are
Fig. 3. Blood concentrations of unchanged compound (A), monomethylaminc
(^), or total radioactivity (O) for typical animals as a function of time after a not unexpected, since 5 times as much of a dose of MA is
single administration of |'4C]NDMA by gavage to a male Fischer 344 rat at a
dose of 14 fimol/kg (A) or a single administration of [uC]NDMA-rf6 by gavage to
excreted unchanged in the urine if the molecule is deuterated
another rat at a dose of 15 jimol/kg (B). than if it is not (15). Equilibrium dialysis revealed negligible
plasma protein binding for NDMA, as reported previously (22),
and also for NDMA-¿6.
enon is due to the much slower rates of both formation and
elimination of MA-rf, caused by the deuterium isotope effects DISCUSSION
on both processes.
The effects of complete deuterium substitution on the toxi- The results presented in this article extend the original dis
cokinetics of NDMA in the rat have been studied previously covery of monomethylamine as a urinary metabolite of NDMA
(18). However, in the earlier study large numbers of animals in vivo in the rat by Heath and Dutton (25) by confirming its
were used, with a group of rats being killed at each time point presence in the blood as well as urine following i.v. or i.g.
to determine the concentration of NDMA in the blood. It was administration of NDMA. Since MA has been identified as a
considered essential to the present study to redetermine the product of NDMA denitrosation by microsomes from the livers
toxicokinetic parameters for NDMA at the same time as MA, of acetone induced rats in a reaction catalyzed by the same form
using serial blood sampling to obtain a whole set of blood of cytochrome P450 thought to be responsible for much of the
concentration versus time curves from each single animal and NDMA metabolism that occurs in vivo (6), it can be inferred
hence avoid the large interanimal variation seen in the previous that denitrosation to MA is also a significant elimination route
experiment. Comparison of the results presented in Table 1 to for NDMA in the intact rat.
those previously published (18) revealed a number of differ Determination of the fraction (/„,)of an i.v. bolus dose of
ences, most notably that of Kss for which the values in Table 1 NDMA that is ultimately metabolized by the denitrosation
are approximately twice those previously reported, and since pathway (i.e., to MA) can be accomplished by using the equation
those in the present study are also equivalent to total body /„= (AUC(m).,vx dose„.i.,.)/(AUC„.i...
x dose,.,,.)
water (19) they would seem to be more reliable. It is frequently
difficult to interpret the values for isotope effects obtained where AUCm.,g.is the area under the blood concentration versus
under even the most favorable circumstances, such as using time curve for MA measured following an i.g. dose (dosera,¡.g.)
pure enzymes in vitro. Consequently, the results of attempts to of MA, and for which parameters mean values have been
calculate the isotope effect for a particular reaction in the intact previously calculated in this rat model to be 1.06 /¿mol•¿
min/ml
animal must be treated extremely cautiously. Nevertheless, we and 81.9 jumol/kg, respectively ( 15). AUC(m).¡.,.
is the area under
made estimations of the isotope effects on various processes the blood concentration versus time curve for MA observed
associated with the metabolism of NDMA along the same lines following an i.v. bolus dose of NDMA (dose,.,v.).
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 IN VIVO DENITROSATION OF A'-NITROSODIMETHVLAMINE

Table 2 Renal excretion following a single i.v. bolus dose off'CJNDMA or f'CJNDMA-dt in the rat
compound"Parameter% Unchanged monomethylamine"NDMA5.14 radioactivity"NDMA3.64

dose excreted in
0-24 h ±0.04 + 0.23 1.98' ±0.05 ±0.25"1
24-48 h ND ND 0.41 ±0.10 0.29 + 0.11 0.80 ±0.05 1.34 ±0.05''
48-72 h ND ND 0.09 ±0.02 0.02 + 0.01' 0.79 ±0.15 0.64 ±0.06
hRenal
0-72 ±0.040.031
0.11
NDNDNDMA-i/60.11 12.90± \.96fTotal
5.63 ±0.28NDMA-</612.60± 11.71 ±0.25''
5.22 ±0.17NDMA-</69.74

blood clearance ±0.010Metabolic

(ml/min/kg)NDMAND*
" A dose of 14.1 ±0.4 ¿imol/kgof [I4C]NDMA was given to a group of 4 male F344 rats weighing 196 ±4 g and a dose of 6.36 ±0.54 jimol/kg of ['"CJNDMA-
dt was given to another group of 4 rats weighing 195 ±18 g. Results are presented as mean + SE.
* ND, not detected.
c P < 0.01. significantly different from the group of rats receiving NDMA (Student's t test).
* P< 0.001, significantly different from the group of rats receiving NDMA (Student's t test).
* P< 0.05, significantly different from the group of rats receiving NDMA (Student's t test).
f P < 0.02, significantly different from the group of rats receiving NDMA (Student's / test).

The theory (26) is that assuming MA is formed from NDMA
exclusively in the liver (22), if preformed MA is administered
as an i.g. dose it will all enter the body through the liver and,
consequently, the subsequent elimination will simulate the fate
of MA which is formed inside the liver as a metabolite of
NDMA. Therefore, by comparison of the AUC values it should
be possible to infer the total quantity of MA that is formed
from NDMA (including any which is metabolized even before
leaving the liver). A number of assumptions must be made for
this calculation to be valid: (a) the metabolism must occur
exclusively in the liver, a thesis which has been supported
experimentally (22); (b) MA must be completely absorbed from
the intestine after i.g. administration and reach the liver intact.
We have previously demonstrated (15) that MA is almost
completely absorbed and while we cannot entirely rule out
metabolism in the intestines, we think that it is unlikely to be
extensive; (c) the administered MA must partition into the
hepatocyte to the same extent as if it had been formed there.
As judged by the large apparent volume of distribution of MA
(15), it is unlikely that there would be any significant barriers
for the entry of MA into liver cells. It has also been reported
that MA is accumulated by isolated rat hepatocytes to concen
trations higher than those in the extracellular medium (27); (d)
the ¡mine,which is the proposed intermediate between NDMA
and MA (Ref. 16; Fig. 1) must be assumed to be completely
converted to MA; (e) saturation must not occur in any of the
processes.
Application of the above equation to the individual sets of
values used to construct Table 1 led to the/m values given under
Method 1 in Table 3. In the case of NDMA, denitrosation, as
determined from the fraction metabolized to MA, appears to
account for about one-fifth of the total elimination in the
normal adult male Fischer 344 rat. This number is similar to
the 9-15% denitrosation reported previously in studies using
ethanol or acetone induced microsomes (4). If one assumes that
the induction process merely increases the amount of enzyme
which metabolizes NDMA to an intermediate common to both
pathways, then induction should increase only the rate of prod
uct formation and not the relative proportions of the products
(6). Consequently, the in vivo results appear to lend support for
the involvement of the same enzyme system as that involved in
vitro, namely cytochrome P450IIE1.
Surprisingly, the mean extent of in vivo NDMA-i/6 denitro
sation (39.8%), when calculated by substituting into the above
equation the mean values for AUCm,¡.g. and dose m.¡.g.
of 4.45
fitno\-min/ml and 89.6 /¿mol/kg which were previously ob
tained for MA-i/3 (15), was over one-half again greater than
that of NDMA (21.3%). Uncritical application of the two-tailed
Student's / test to the difference between these means gave P =
0.136, but examination of the F statistic revealed considerable
heterogeneity of variance for the NDMA versus NDMA-rf6
groups. Applying a modification of the t test that is generally
considered more appropriate for situations involving unequal
variances (28), P = 0.092 was obtained. Alternatively, the two-
tailed Wilcoxon rank sum test gave P = 0.114. Since the
hypothesis under test was that the deuterated substrate would
be more efficiently denitrosated, i.e., that the difference in
means would be in the direction observed, a one-tailed test
could be justified. If that is done, each of the P values given
above would be halved (to 0.068,0.046, and 0.057, respectively).

Table 3 Contribution of metabolic denitrosation to the total elimination of NDMA and NDMA-dt in various male Fischer rats
NDMA" NDMA-</<,°

Rat Method 1 Method 2 Rat Method 1 Method 2

01.1892
0.2113
0.2014
0.251Mean

+ SE 0.213 ±0.0130.1290.1440.1370.1710.1
45 ±0.009r2'3'4'5'6'Mean + SE0.5320.3350.1940.2380.3160.7740.398
±0.0890.6450.4060.2350.2890.3830.9390.483
±0.108
" Values given for the individual rats are of fraction metabolized to MA.
Method 1 uses the equation

AUC,„ x dose»,
AUCm.¡...
x dose,.

Method 2 uses the equation

fm ~
AUC,,,.i... x dose,.,...
AUCm.¡... x dosc,.¡...
1148

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 IN VIVO DENITROSATION
Calculations of/m based on the former equation assume that
metabolism of MA takes place in the liver. However, the sites
of MA biotransformation are not yet definitely determined (15)
and, if no hepatic metabolism of MA occurs, then all of the
MA that is formed from NDMA in the liver should be released
into the systemic circulation, assuming that biliary excretion is
negligible (15). Consequently, an alternative approach to the
calculation of/,, would be to use
/„,= (AUC,»,.¡.,.x dosem.¡.v.)/(AUCm.¡.v.
x dose,.¡, )
where AUCm.¡.v. is the area under the blood concentration versus
time curve for MA measured following an i.v. bolus dose of
MA (dosem.¡.,.). The solution of the latter equation would give
values of/,, that do not rely on the assumptions of complete
absorption and delivery of unchanged MA to the liver following
an i.g. dose but may be underestimates of the true numbers.
The calculations were carried out by substituting into the latter
equation values for AUCm.¡.v. and dosem.¡.».
which had been
determined previously to be 358 nmol-min/ml and 18.9 /¿mol/
kg for MA or 144 nmol-min/ml and 3.52 /¿mol/kgfor MA-rf,,
respectively (15). The results presented in Table 3 under
Method 2 reveal an even greater difference in the fraction
metabolized to MA between NDMA and NDMA-</6 than was
indicated using Method 1, with mean values of 14.590 and
48.3%, respectively. Statistical analyses similar to those used
above demonstrated P values of 0.037, 0.026. and 0.010 for the
two-tailed t tests with equal and unequal variances and the two-
tailed Wilcoxon rank sum test, respectively. Again, making the
assumption that the difference in the means would be in the
direction observed and applying one-tailed tests produced P
values of 0.019, 0.013, and 0.005, respectively.
While the most appropriate statistical test (and, therefore,
the level of statistical significance) for assessing the difference
between the NDMA and NDMA-</6 groups in the fraction
metabolized to MA is not clear, the results nevertheless raise
the very interesting conclusion that a chemical modification as
subtle as replacing all of the hydrogen atoms of the substrate
with a heavier isotope is capable of perceptibly increasing the
importance of the metabolic denitrosation pathway with respect
to the competing dealkylation route. This is interesting because
the in vitro isotope effects on the competing metabolic pathways
were the same in the presence of microsomes from the livers of
acetone-induced rats, indicating that the balance between them
was not affected by substrate deuteration (6). There could be a
number of reasons for such in vivo differences. Even if it is
possible to assume that all of the metabolism of NDMA takes
place in the liver (22), some of the metabolizing capacity may
reside in the nonmicrosomal fractions of the cell, or in micro-
somal enzyme systems other than cytochrome P450. In addi
tion, since the microsomal experiments were carried out with
induced animals, the induction may have increased certain
enzyme systems (e.g., P450IIE1) selectively and hence effec
tively masked others (i.e., other forms of P450 or other en
zymes).
In any event, an increase in the fraction of NDMA denitro-
sated to MA upon deuteration would be consistent with the
previously reported significantly lower incidence of hepatic
tumor formation in rats dosed with NDMA-i/6 (14) and the
lower levels of alkylated bases found in the livers of rats dosed
with NDMA-i/6 in comparison to equivalent doses of NDMA
(23). Both of these experiments suggest the existence of some
diversionary elimination pathway or pathways other than me
tabolism to ultimately carcinogenic alkylating species. The
0.1% of a dose of NDMA-rf6 accounted for by urinary excretion
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OF JV-NITROSODIMETHYLAM1NE
of the unchanged compound is too small to contribute signifi
cantly to the alternative elimination routes, but perhaps the
slower rate of metabolism of the deuterated compound would
allow repair mechanisms to cope better with the damage. An
increase in the proportion of a dose of NDMA traversing the
denitrosation pathway could account for the differences since
it separates the nitrogens of the /V-nitroso group and hence
precludes conversion of the NDMA to the alkylating diazonium
ion.
In the case of a compound such as NDMA which is not
bound to plasma protein and which is metabolized solely and
almost completely in the liver, when the substrate concentration
at the active site of the enzyme is low compared to the A'm(i.e.,
first order toxicokinetics as in the experiments presented here),
Cl, approximates Vmm/Km (18). However, the hepatic micro
somal values of Km and Vmaxfor the metabolism of NDMA by
noninduced rat preparations have been extensively studied in
vitro (29, 30). In the past, the suggestion has been put forward
to use the data obtained for microsomal incubations of a number
of drugs to calculate their Cl¡values and then to verify these
calculations by determining the same parameter in isolated
perfused livers (31). In the case of NDMA, if it is correct that
all of the metabolism of the compound that occurs in the body
takes place in the liver, then the study could be extended one
step further to the intact animal. If one takes the recently
published value of 1.6 nmol/min/mg of microsomal protein for
Vmaxin microsomes from noninduced rats (29), multiplies by
50 (31) to give the metabolic rate per g of liver and. assuming
that the liver accounts for 5% of the total body weight (32),
converts the value to a rate per kg basis, one obtains a figure
for Vmaxof 4000 nmol/min/kg. A recent estimate of the A'mof
the high affinity, low capacity enzyme which would be respon
sible for almost all of the NDMA metabolism at low concen
trations was reported to be in the range 20 to 30 /¿M
(30), i.e.,
20 to 30 nmol/ml. Calculation of the Vmax/A'n,ratio (i.e., Clt)
from these microsomal parameters gives a value of 133 to 200
ml/min/kg. While this value is lower than that derived for Cl,
from our present in vivo studies (388 nmol/min/kg), the differ
ence may be explained by the observation that the formaldehyde
production from isolated rat liver microsomes was only about
one-half of that present in the corresponding whole liver ho-
mogenates or 9000 x g supernatant fractions (33). Loss of
enzymatic activity upon isolation of microsomal fractions ap
pears to be a widespread finding for cytochrome P-450-me-
diated reactions and it has been attributed at least in part to a
greater susceptibility to lipid peroxidation in the microsomal
membranes (34). In any event, our estimate is larger than
numbers produced by similar calculations from data derived
from rat liver slices or isolated perfused rat livers which when
converted to the same units approximated 36 and 72 nmol/
min/kg. respectively (32, 35).
If the microsomal data can be considered a reasonable pre
dictor of the in vivo Cl¡in the rat, similar calculations for the
human may be valuable in assessing the risks of nitrosamine
exposure. Values have already been published for the Michaelis-
Menten constants for the high affinity enzyme in human micro
somes (36). The mean values for A'mand Vmaxwere 36 JIMand
1.5 nmol/min/mg of microsomal protein, respectively. A simi
lar calculation of Cl, for the human yields a value of approxi
mately 200 ml/min/kg, if a factor of 2 is included for the loss
of activity brought about by isolating the microsomes. This
latter value translates to a hepatic blood clearance of about 19
ml/min/kg which is close to the hepatic blood flow rate (Q) of
1149
 IM VIVO DENITROSATION
21 ml/min/kg for the average 70-kg human, using the equation
C/H = QCl,/(Q + C/,)
Consequently, the liver would be expected to metabolize
NDMA to a large extent with an extraction ratio (Ciu/Q) of
approximately 90%, meaning that only about 10% of the nitro-
samine entering the body via the gastrointestinal tract passes
the liver and reaches the systemic circulation.
Extrapolations from animals to humans may prove to be
important, since NDMA has been confirmed as a frequently
observable constituent of normal human urine (37) and may
thus pose a significant cancer risk for people. If an enzyme
capable of selectively denitrosating this potent carcinogen can
be sufficiently induced by some innocuous means, it might be
possible to protect the body from exposure to NDMA even
when the carcinogen is endogenously produced and exposure is
unavoidable.
ACKNOWLEDGMENTS
We would like to thank Dan Logsdon for expert technical assistance,
Charles Riggs for statistical evaluation, and Dr. Lucy Anderson for
helpful discussions.
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1150
Metabolic Denitrosation of N-Nitrosodimethylamine in Vivo in
the Rat
Anthony J. Streeter, Raymond W. Nims, Pamela R. Sheffels, et al.

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