[CANCER
RESEARCH
28, 642-652, April 1968]
The in Vivo Binding of ß-Propiolactone to Mouse Skin
DNA, RNA, and Protein1
N. H. Colburn and R. K. Boutwell
McArdle Laboratory
¡orCancer Research, University
of Wisconsin Medical Center, Madison, Wisconsin 63706
SUMMARY
The binding of tritium-labeled /3-propiolactone to mouse
skin DNA, RNA, and protein was investigated. Binding of the
lactone to RNA and protein, as well as to DNA, was observed.
When propiolactone dose or mouse susceptibility was varied,
the binding to skin DNA, RNA, and protein was found to
correlate with initiation of tumorigenesis. The maximum bind
ing of /3-propiolactone-3H to DNA, RNA, and protein was
attained at 2-12 hours after treatment. The rate of decay of
specific activity of /3-propiolactone-3H-DNA in vivo was faster
than either the rate of metabolic turnover or the rate of in
vitro depurination. The rate of decay of specific activity of
/3-propiolactone-3H-RNA in vivo was faster than the rate of
metabolic turnover. Autoradiographs of mouse skin at 2.5 hours
after treatment with tritiated propiolactone showed heavy
labeling of cornified epithelium and hair follicles with less
labeling of basal cells. Hydrolysis and chromatography of skin
RNA after treatment with tritiated /3-propiolactone showed
that the major binding product was 7-(2-carboxyethyl)guanine.
The data are discussed with respect to the possible significance
in carcinogenesis of the formation of 7-(2-carboxyethyl)-guanine
in RNA and DNA.
INTRODUCTION
BPL2 has been shown to be carcinogenic for the skin of rats
(13, 35), mice (33), and more recently for guinea pigs (27)
and golden hamsters (28). The lactone also initiates the for
mation of skin tumors in mice (8, 34). We previously reported
the binding of /3-propiolactone to mouse skin DNA in vivo
and its correlation with initiation of papilloma formation when
BPL dose or line of mice was varied (8). It was shown that
BPL reacts with skin DNA in vivo to form 7-(2-carboxyethyl)guanine, in analogy with the in vivo reactions of other
alkylating agents with nucleic acids. The present report is con
cerned with the binding of tritiated BPL to skin protein, RNA,
and DNA, and the possible correlations with initiating ability.
1 This work was supported in part by grants from the American
Cancer Society (E-6), the Alexander and Margaret Stewart Trust
Fund, and the USPHS (CRTY-5002).
2 Abbreviations used are : AAF, 2-acetylaminofluorene ; BPL,
/î-propiolactone; DAB, dimethylaminoazobenzene
; DMN, dimethylnitrosamine.
Received September 11, 1967; accepted December 20, 1967.
642
Covalent binding to protein and both nucleic acids in vivo
has been found for most carcinogens tested (26). Thus, for
most carcinogen.--, binding to any one of these three macromolecules could be significant in carcinogenesis. In the case
of the liver carcinogen AAF, both RNA and protein binding
have been shown to correlate in extent with tumorigenesis
when species susceptibility was varied (25). Magee and Farber
(24) demonstrated that RNA methylation by DMN gives such
a correlation when organ susceptibility is varied. Davenport
et al. (12) found a correlation with carcinogenicity of aromatic
hydrocarbon binding to skin "h-like" proteins. Brookes and
Lawley (5) demonstrated a correlation of mouse skin DNA
binding and not RNA or protein binding for a series of aro
matic hydrocarbons when Iball's index was used as a measure
of carcinogenic potency. Roberts and Warwick (31) reported
for DAB the correlation of ribosomal RNA binding, but not
that of protein or DNA, with carcinogenesis when tissue or
species was varied. In the studies described above, the max
imum binding, occurring at times of the order of 12 to 24 hr
after administration of the carcinogen, was determined. When
Warwick and Roberts (40) determined binding at times of 1
to 3 months, it was the DNA binding which correlated with
carcinogenesis, with a level equal to half the 2-day value, while
RNA and protein contained no bound DAB-tritium at these
times. Ethylation of liver RNA by the carcinogen ethionine
correlates, but protein incorporation fails to correlate, with
tissue susceptibility (14). Ethionine appears to be a notable
exception to the generalization that carcinogens bind to both
nucleic acids. Farber (15) reported that ethionine shows little
or no binding to DNA.
Thus there is suggestive evidence against a role for protein
binding in the case of some carcinogens, against a role for RNA
binding in the case of some carcinogens, and against a role for
DNA binding in the case of ethionine. The only clear pattern
that appears to be emerging is that binding to one or both
nucleic acids has consistently shown positive correlation with
tumorigenesis.
In the present study, and in others to be reported elsewhere,
we have further examined the significance of BPL binding to
skin DNA, RNA, and protein. We have looked for possible
correlations of macromolecular binding and tumorigenesis
through introducing the following variables: (a) BPL dose,
(£>)
line of mice, (c) time of determination of binding, and (d)
structural variation among related compounds. As another ap
proach to examining the significance of macromolecular binding,
we have characterized some of the binding products and tested
CANCER RESEARCH VOL. 28
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ß-Propiolactone Binding
for some of the functional consequences which might be pre
dicted to result from the particular structural alterations found.
The present report concerns the extent and nature of binding
of BPL-:!H to skin proteins and nucleic acids, the kinetics of
binding, the distribution of bound BPL-3H in skin, and com
parative binding and tumor studies using mice bred for sen
sitivity or resistance to BPL.
MATERIALS AND METHODS
Materials
/3-propiolactone was supplied by Testagar and Company,
greater than 99% pure. BPL was used directly from freshly
opened ampules, or redistilled at 11 mm Hg and 51°C.Croton
oil was obtained from S. B. Penick and Co. and used without
further purification. Tritiated /3-propiolactone was obtained
from Nuclear Chicago at a specific activity of 167-288 me/
mmole and greater than 98% radiochemical purity when
freshly prepared. The purity was rechecked periodically by
reverse isotope dilution analysis involving distillation at 11 mm
of mercury and 51 °C.The BPL-3H remained greater than 90%
radiochemically pure for three months after preparation; nor
mally it was used within three months after preparation.
Binding and Tumor Experiments
BPL or BPL-3H was applied by syringe to the shaved backs
of mice as a freshly prepared solution in anhydrous acetone
containing 120, 240, or 480 wnoles in 0.3 ml per mouse. The
croton oil used in tumor experiments was applied twice weekly
as two drops (50 /*!) in a 0.5% solution in benzene.
Female skin tumor susceptible (STS) mice supplied by A. R.
Schmidt Company and originally bred by Boutwell (2) were
used for all experiments unless otherwise specified. Usually the
mice used were 7 to 8 weeks old and weighed 25 to 30 grams.
An area of about 15 sq cm on the back was shaved.
STS females and males were challenged with 480 Amóles/
mouse of BPL, followed at two weeks by repeated applications
of croton oil. At twelve weeks after the BPL treatment, the
male and female mice which had the largest numbers of papillomas (normally those with at least 4/mouse) were bred to
gether and those with no papillomas were bred. Offspring were
called first generation. These offspring were then challenged.
The first generation sensitive mice which had the most tumors
were then bred and the first generation resistant mice which
had no tumors were bred, to give a 2nd generation.
Extraction of DNA, RNA, and Protein from Mouse Skin
Chemical Procedures. Except when otherwise specified,
DNA, RNA, and protein were extracted according to a modifi
cation of the procedure of Kirby (19) which fo'lows. Treated
skins of four to six mice were frozen in liquid nitrogen, scraped,
and pulverized as described previously (8). The pulverized
skin was homogenized in the cold in 4 to 6 ml of 5% p-aminosalicylate-1% sodium dodecyl sulfate. The aqueous layer was
then extracted 3 times with one volume of a phenol :8-hydroxyquinoline:m-cresol:water
mixture (500:0.5:70:55 by weight)
to remove protein. DNA was precipitated from the aqueous
phase with one volume of cold ethoxyethanol and dissolved in
0.001 M K2HPO4 buffer (pH 7). Two volumes of cold ethanol
APRIL 1968
were added to the remaining aqueous phase to precipitate RNA.
The DNA solution was incubated 15 minutes with RNase,
then made l M in NaCl, treated with phenol, precipitated, and
redissolved in buffer. One-half volume of hexadecyltrimethylammonium bromide was added to form the water-insoluble
salt of the DNA. The DNA salt was then washed thoroughly
with water, followed by several washes with 70% ethanol con
taining 2% sodium acetate to convert the DNA to the sodium
salt. The RNA was converted to the hexadecyltrimethylammonium salt and washed by the same procedure as the DNA.
Each nucleic acid was finally dissolved in dilute standard saline
citrate (0.015 M NaCl:0.0015 M trisodium citrate). The protein
in the combined phenol layers was precipitated by addition
of the phenol layer to a large volume of cold methanol. The
mixture was centrifuged and the protein was washed (1-2 ml
volumes) once with methanol, once with methanol :ether (1:1),
4 times with 100% ethanol, and with anhydrous ether until
dry. Typical yields/5 skins: DNA, l mg; RNA, l mg; pro
tein, 200 mg.
When indicated, a modified Marmur procedure was followed
according to the steps previously described (8).
The Somerville-Heidelberger procedure for extraction and
purification of soluble and insoluble skin protein was performed
according to the acetone precipitation method of Somerville
and Heidelberger (36) with no modifications.
Determination of Specific Activity. The DNA solutions were
hydrolyzed with deoxyribonuclease, determined by O.D. at
1%
260 nv using an E . of 280, and periodically confirmed by
1cm
diphenylamine determination. Aliquots of the DNA solution
were counted in a liquid scintillation spectrometer in 10 ml of
ANPO (a medium containing 295.2 gm of naphthalene, 18.4
gm of 2,5-diphenyloxazoIe, 0.1839 gm of «-naphthylphenyloxazole, 1400 ml of xylene, 1400 ml of dioxane, and 840 ml of
ethanol). The RNA solutions were hydrolyzed with ribonu1*
clease, determined by O.D. at 260 m/* using an E t of 220,
i cm
and periodically confirmed by orcinol determination. Aliquots
of the RNA solution were counted in the liquid scintillation
spectrometer in ANPO. Protein was hydrolyzed 30 min at 80°C
in 0.5 N NaOH and determined by the Lowry procedure (23)
using bovine serum albumin standard; the hydrolysate was
counted in the liquid scintillation spectrometer. In a typical
experiment, the nucleic acid solutions were counted at 100-500
counts per minute above background, and the proteins were
counted at 500-1000 counts per minute above background. To
convert specific activity in dpm/gm to wnoles BPL bound/gm
or mmoles BPL bound/mole nucleic acid-P, 25 n\ aliquots of
the solutions of BPL-3H applied to the mice were diluted to
appropriate volumes with water and counted to determine the
specific activity of the BPL-3H applied in dpm/wnole.
Purity of Macromolecules Extracted. DNA and RNA sam
ples obtained by the modified Kirby procedure were tested by
the method of Keeler (18) for protein content. DNA samples
were found to have protein contents of 3% or less in 2 deter
minations. RNA samples were found to contain less than 3%
protein in 2 determinations. Four orcinol determinations of
DNA samples showed DNA to contain less than 2% RNA.
643
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N. H. Colburn and R. K. Boutwell
Hydrolysis and Chromatography of BPL-3H-RNA and Lo
cation of Activity. Hydrolysis and paper chromatography in
two dimensions of BPL-3H-RNA was performed just as pre
viously described for DNA (8). The chromatogram was cut
into rectangles (1 x 1.5 inches), the rectangles were cut into
strips, and strips from each rectangle were eluted with 1.5 ml
of water at 37°C for 16 hr and washed twice with 0.5 ml
0.01 M NH4OH. The combined eluates were evaporated to dryness, the residue was dissolved in 0.01 M NH4OH (500 M!),
and 450 M!were counted.
Determination of in Vitro Tritium Loss from in Vitroalkylated BPL-3H-DNA. Calf thymus DNA (Worthington) (5
mg per 10 ml 0.03 M K2HP04, pH 7.3) was reacted with 400
emoles (25 M!) of BPL-3H containing 51 MCat 25°C for 20
minutes. The solution was then twice dialyzed against 2 liters
of the buffer at 4°C(24 hr total). An aliquot of this BPL-3HDNA solution was used as the zero-time sample, and the rest
of the solution was incubated at 37°C with a few drops of
chloroform added. Aliquots were removed at various times up
to 180 hours after zero time and dialyzed in the cold. The
specific activity of the DNA was determined by counting DNA
CD
200
400
600
800
1000
BPL DOSE (^MOLES/MOUSE)
Chart 1. The binding of /î-propiolactone-3H to mouse skin DNA,
RNA, and protein as a function of 0-propiolactone
(BPL) dose.
Tritiated BPL was applied once to the skin of skin tumorsusceptible mice in 03 ml of acetone solution. Mice were killed
and skins taken at 4 hr after BPL application. •DNA, O RNA,
A protein; nucleic acid-P, nucleic acid phosphate.
644
solutions in ANPO and determining concentrations by O.D. at
1%
260 m/t using an E.
value of 156.
lem
Autoradiography
of BPL-3H-treated
Skin Samples.
One
mouse received 480 /¿molesof BPL-3H containing 500 MC.Four
biopsies of 1 sq cm were taken from nonadjacent areas of the
skin at times from 2.5 to 44 hr after BPL-3H application. The
biopsies were fixed in formalin and submitted to autoradiography by the method of Lesher et al. (22).
RESULTS
Binding of BPL-3H to Mouse Skin DNA, RNA, and Protein.
Mice given zero to 960 ¿¿moles
BPL-3H as described in Ma
terials and Methods were killed at 4 hours after treatment and
the specific activities of the extracted DNA, RNA, and protein
were determined. Chart 1 shows that binding is linear up to
480 wnoles and that saturation appears to occur at about 480
/»molesper mouse for RNA and protein, as well as for DNA
shown here and reported previously (8). We have found a
saturation at the 480-Mmole dose and proportional effect below
this dose for tumors (papillomas per mouse) arising from a
single initiating dose of BPL followed by croton oil as promoter
(8). Thus BPL-3H binding to all 3 macromolecules studied
correlates with papillomas per mouse produced as a function
of dose.
It can be seen that the RNA binding is somewhat higher
than the DNA binding and that the protein binding is about
twice as high. The BPL to DNA or RNA binding using 480
Minólescorresponds to about 1 mmole BPL/mole DNA or RNA
phosphate.
Kinetics of Binding of BPL-3H to Skin DNA, RNA, and
Protein. Mice which received 480 MinólesBPL-3H were killed
at various times afterward and the specific activities of the ex
tracted DNA, RNA, and protein were determined. Chart 2
shows the binding as a percent of the 2-hour value plotted
against time of determination. Each symbol represents data
from one experiment. The DNA binding appears to remain
constant from 2 to 12 hours, drops sharply to 50% of the
2-hour value at 24 hours, then decreases at a slower but con
stant rate through 96 hours. The RNA binding decreases slowly
through the first 12 hours, then faster at a constant rate to
about 20% of the 2-hour value at 96 hours. The protein binding
appears to decrease faster during the first 24 hours than sub
sequently, reaching about 50% of the 2-hour value at 96 hours.
Half-lives for loss of radioactivity calculated from the final
slopes of the log plots (Chart 3) are as follows: DNA, 72
hours; RNA, 39 hours; and protein, 125 hours. The loss of
DNA-specific activity after BPL-3H alkylation in vivo may be
occurring by at least two possible mechanisms. These include
the turnover of DNA and the loss of 7-(2-carboxyethyl)guanine, due to the labilization of the N-9 position by N-7 alkyla
tion. Hennings and Boutwell3 have found that the specific
activity of mouse skin DNA containing thymidine-3H intro
duced by prelabeling decreases with a half-life of about 120
hr. Moreover, the rate of DNA synthesis after treatment with
480 Minólesof BPL remains below 50% of the control value
from 2 through 10 hours after BPL treatment, then rises above
CANCER RESEARCH VOL. 28
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ß-Propiolactone Binding
32
TIME
(HR)
64
96
Chart 3. Log of the percent of the 2-hr DNA-, RNA-, and
protein bound activity remaining, as a function of time after
tritiated ß-propiolaetone application. Data are taken from Chart
2, by using the average of points shown for given times. —•—
DNA, —O—RNA, —A—protein.
depurination at 37°Cand pH 7.2 were found to be 25 to 200
hours for these alkylating agents, depending on the compound
used. Loss of specific activity of BPL-3H-DNA incubated in
vitro has been determined, as illustrated in Chart 4. Calf thy
mus DNA which had been reacted ¿nvitro with BPL-3H was
dialyzed in the cold to remove unreacted BPL-3H, then in
cubated at 37°Cfor a week at pH 7.3. Aliquots of the solution
40
60
TIME (HR)
Chart 2. Time course of binding of tritiated ¿8-propiolactone
(BPL-3H) to skin DNA, RNA, and protein. 480 Amólesof BPL-3H
was applied once to the skin of skin tumor-susceptible
mice in
0.3 ml of acetone solution. Mice were killed and skins taken at
times from 2 to 96 hr after BPL-3H application. Each symbol
represents data from one experiment. The solid circles represent
data for insoluble protein taken from Chart 5.
200% of the control value at 24 hours.3 The early constant
level of specific activity could be partially explained by the
low rate of DNA synthesis during that same time. The rapid
drop in specific activity from 12 to 24 hours could be due to
the large increase in DNA synthesis during that time. The
final rate of specific activity loss from DNA may be partially
due to the normal rate of turnover of DNA.
Lawley and Brookes (21) have shown that N-7 alkylguanines ar<- released at neutral pH from DNA which has been
alkylated with 2-chlorethyl-2-hydroxyethyl
sulfide, methyl
methanesulfonate, or ethyl methanesulfonate. The half-lives for
3 H. Hennings and R. K. Boutwell, unpublished
APRIL
results.
were removed at various intervals and the specific activity of
the DNA was determined. It can be seen that the specific ac
tivity decreased at a constant rate with a half-life of about 150
hours. Hence the specific activity decay after 24 hr of BPL-3HDNA in vivo (72-hr half-life) is probably due to both depur
ination (150-hr half-life) and DNA turnover (120-hr half-life).
The in vivo decrease of RNA specific activity after BPL-8H
alkylation might be occurring by RNA turnover in the skin
or by enzymatic excision. Chemical depurination has been
shown not to occur in polyribonucleotides at neutral pH (3).
However, it is possible that there could be phosphotriester for
mation and consequent chain scission and loss of RNA segments
as discussed by Lawley (20). The half-life of RNA turnover
in the skin has been found by Hennings and Boutwell3 to be
about 100 hours. Perhaps an additional mechanism, such as
enzymatic excision, was occurring to account for the net in
vivo half-life for BPL-3H-RNA of 39 hours.
Warwick and Roberts (40) have recently reported a per
sistent binding of dimethylaminoazobenzene to liver DNA of
50% of the 2-day level, at 3 months after treatment. These
data were interpreted to indicate a lack of repair. This was
in contrast to a complete loss of bound DAB from RNA and
protein in a few weeks. We have previously reported a decrease
of DNA-bound BPL-3H to a few percent of the maximum
value at 2 weeks after treatment (8). We have since tested the
3-week binding of BPL-3H to DNA, RNA, and protein.
Table 1 shows that DNA binding at 3 weeks had decreased
to 0.9% of the 4-hour value, RNA binding had decreased to
0.9%, and protein binding had decreased to 10%. We were
1968
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645
N. H. Colburn and R. K. Boutwell
determining 30-300 counts per minute against a background of
14 counts per minute for these determinations.
Total skin protein contains insoluble keratin and collagen
which would be expected to have a relatively slow rate of
turnover, as well as less stable soluble proteins which include
most enzymes. In order to determine the rate of loss of bound
BPL-3H from soluble protein, we fractionated the skin protein
with isotonic KC1 into KCl-soluble and -insoluble protein and
determined the specific activity of the purified preparations at
various times after treatment. Chart 5 shows the time course
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30
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TIME
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40
60
TIME (HR)
120 150
Chart 4. Hydrolysis at pH 73 and 37°Cof 3H-carboxyethylated
DNA after in vitro reaction of the DNA with tritiated /3-propiolactone (BPL). Experimental details are given in the text. The
upper graph shows the specific activity of BPIWH-alkylated
DNA
as a function of time of incubation at 37°C.The lower graph
shows the DNA specific activity as the log of the r/< of t lie zero
time value. DNA-P, DNA phosphate.
Chart 5. Time course of binding of tritiated /3-propiolactone
(BPL) to KCl-soluble and -insoluble skin protein. Tritiated ßpropiolactone (480 Amóles) was applied once to the skin of skin
tumor-susceptible mice in 0.3 ml of acetone solution. Mice were
killed and skins taken at times from 0 to % hr after BPL appli
cation. Results are expressed as the average binding for 2 sep
arately pooled pairs of skins. The upper graph shows binding for
KCl-insoluble protein, which constitutes over 90% of the total
protein. The lower graph shows binding for soluble protein.
Table 1
Time4hr3
bound'mole
BPL
DNA-P9908.90.9Amóles
boundmole
BPL
RNA-P11009.90.9/¿moles
boundL'i
BPL
nprotein5.90.5910
weeks3-week
binding
as a % of 4-hr
value/tmoles
Binding of /J-propiolactone^H*
to mouse skin in DNA, RNA, and protein at 3 weeks after
BPL-3H application. BPL-3H, tritium-labeled
/3-propiolactone;
DNA-P, DNA phosphate;
RNA-P, RNA phosphate.
0 Each binding value represents data from a pooled group of 4-5 mouse skins.
* Skin tumor-susceptible mice received 480 AmólesBPL-sH/mouse at zero tune.
646
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ß-Propiolactone Binding
of binding of BPL-3H to soluble and insoluble protein, each
point representing the average value for two pairs of skins. It
can be seen that the two fractions show time courses of binding
similar to each other and to total protein obtained by the
phenol method, as well as similar absolute binding in /»moles/
gm.
Zero-time Binding. It can be seen in Chart 5 that protein
shows a high zero-time BPL-3H binding, similar to the case
reported previously for DNA (8). Table 2 shows values for
zero-time binding obtained for DNA, RNA, and protein using
the Kirby procedure of extraction. Values are expressed as the
mean of two experiments in mmoles BPL/mole phosphate or
/»molesBPL/gm ±the average deviation from the mean. Zerotime binding is clearly higher than that for subsequent times in
the case of DNA, RNA, and protein.
Since zero-time binding is high, the question was asked
whether binding measured at two hours or later is occurring
in vitro. To answer this question for DNA, nonradioactive BPL
was added to the homogenization medium (Marmur procedure)
for pulverized skins taken at zero time, 2 hr, 4 hr, and 24 hr
after BPL-:!H treatment. If in vitro binding is occurring, the
addition of unlabeled BPL to dilute any labeled BPL present
should diminish the binding of tritiated BPL to DNA. Five
mmoles of unlabeled BPL were added to the homogenization
medium for 5 skins, or 2.1 times the number of Mmolesapplied.
Shown in Table 3 are values for binding in mmoles/mole
DNA-P for BPL-3H at various times after application, with
Table 2
mmoles
boundmole
BPL
DNA-P1.94
boundmole
BPL
P2.57 RNA-
boundgm
BPL
protein12.8
±0.13«mmoles
±3.9
±0.40/¿moles
Zero time binding of /S-propiolactone-3!!6 to mouse skin DNA,
RNA, and protein. BPL-3H, tritiated /J-propiolactone ; DNA-P,
DNA phosphate, RNA-P, RNA phosphate.
a Each binding value represents the mean for two experiments
± the average deviation from the mean. Four to 5 mouse skins
were pooled for each experiment.
b Skin tumor-susceptible
mice received 480 /»moles BPL-3H/
and without the added unlabeled BPL during homogenization.
Binding was calculated in the normal way, using specific ac
tivity determined for applied BPL-3H dose to convert dpm/
Mg DNA to mmoles/mole DNA-P. Since only the zero-time
binding of BPL-3H, and not binding at subsequent times, was
diminished by addition of unlabeled BPL, it is concluded that
binding occurs in vitro only in the case of the zero-time group
and that binding measured at subsequent times is occurring
in vivo.
Distribution of BPL-3H in Mouse Skin. In an attempt to
obtain an estimate of the distribution of BPL in mouse skin
cells, within epidermis, within dermis, and in epidermis as com
pared to dermis, BPL-3H was applied to a shaved mouse skin,
the skin removed, fixed in formalin, then subjected to autoradiography. Fig. 1 shows an autoradiograph of skin taken at
2.5 hr after BPL-3H treatment. The coraified layer of the
epithelium and the hair follicle showed a consistently high de
gree of labeling. Some basal cells appeared to be labeled, but
at a much lower level. Autoradiographs taken at times from
18 to 44 hr showed a similar pattern but with less total la
beling. It was not possible to obtain a quantitative estimate of
the proportion of basal cells or dermal cells labeled.
Since we were unable, with autoradiography, to obtain an
estimate of what percent of the skin cells were labeled with
BPL-3H, we attempted another approach to the same question.
Mice were pretreated with a saturating dose (480 /»molesper
mouse) of unlabeled BPL. At 2 hours (to allow maximum
binding of the pretreatment dose), 240 /uñólesof BPL-3H
were applied. Binding to DNA, RNA, and protein at 2 hours
after the BPL-3H application was assayed. It was reasoned
that if the initial saturation by the 480 /¿molesBPL involved
permeation of nearly 100% of the cells, then the subsequent
binding of the BPL-3H should be blocked. But if the initial
saturation involved only a few percent of the cells and labeling
was random, then the subsequent binding of the BPL-3H
should not be blocked. The results indicate (Chart 6) that
BPL pretreatment using a saturating dose does not depress
CONTROL
DNAOR
" RNA
BPL-PRETREATED
DNA
RNA
PROTEIN
PROTEIN
Table 3
DNA-P)-Time
BPL-3H«binding (mmoles/mole
BPL' during
homogenization0.8501.121.000.558
(hr)Zero2424Control2.061.121.020.462+
Effect of addition of unlabeled BPL to homogenization me
dium, on binding of BPL-3H to mouse skin DNA. BPL, /3-propiolactone; DNA-P, DNA phosphate.
a Each binding value represents data taken from a pooled group
of 4-6 mouse skins.
6 5 mmoles of unlabeled BPL were added to the homogeniza
tion medium for 5 skins.
c Skin tumor-susceptible
mice received 480 /¿moles BPL-3H/
mouse at zero time. DNA binding was determined for skins taken
at zero time, 2, 4, and 24 hr.
APRIL
1968
Chart 6. The effect of pretreatment with unlabeled /3-propiolactone on the 2-hr binding of subsequently applied tritiated BPL
to skin DNA, RNA, and protein. The pretreatment dose of BPL
was 480 /imoles/mouse. At 2 hr following this application, 240
/¿moles/mouse of BPL-3H were applied. Binding was determined
at 2 hr following the BPL-3H application.
647
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N. H. Colburn and R. K. Boutwell
the binding of subsequently applied BPL-3H to DNA, RNA, or
protein. Hence the results are compatible with the initial sat
uration involving only a few percent of the cells.
Comparison of BPL-3H Binding in Resistant versus Sensi
tive Mice. In order to obtain mice which would give a higher
6.05.0Luo^Q<A\ièU.0D1.00Pa/K/IOUSEZ40.P
PROTSTSk
DNA
RNA
MOLES.RESBSENS3TS••S\lOOhalLLJ0
MOLES
075
\LU¿0.50ESZ
ftd1i.o
'ÃŒRESSTSTRES_3.0-g2-0
tumor yield from a saturating dose of BPL, and also to obtain
mice differing in BPL sensitivity suitable for comparison of
nucleic acid and protein binding, STS mice were bred for sen
sitivity or resistance to BPL. Data presently available on tumor
incidence and BPL-3H binding for the second generation are
shown in Chart 7. This shows that when an initiating dose of
240 wnoles of BPL per mouse was used, followed by repeated
croton oil application, the resistant line gave a tumor incidence
about 10% that of the sensitive line and 25% that of the STS
line. The data indicate that BPL-3H binding to DNA, RNA,
and protein showed correlation with skin tumor susceptibility.
Hence these experiments give no suggestion as to which, if any,
2400
?ozm0
0.25Zm--BINDING240>j
RES,T,
CEG
2000
1600
Chart 7. Comparison of tritiated /3-propiolactone binding and
tumor incidence in female skin tumor-susceptible mice and female
mice bred for sensitivity or resistance to /3-propiolactone. Tumor
incidence is shown in papillomas/mouse
at 13 weeks after treat
ment with 240 Amóles/8-propiolactone/mouse
followed by repeated
croton oil treatment. Tritiated /3-propiolactone binding is shown
for DNA, RNA, and protein extracted 4 hr after treatment of
skins with 240 ¿imolestritiated /3-propiolactone/mouse.
Pa/mouse,
Papillomas/mouse;
RES, second generation resistant line; STS,
skin tumor-susceptible
line; SENS, second generation sensitive
line; BPL, /3-propiolactone.
C 1200
Ì
80O
400
PYNT
ORIGIN
L-JO
ORIGIN G CEG PyNT
SF
G
A
Chart 9. Radioactive
profile of paper chromatogram
from
hydrolysate of radioactive mouse skin RNA (125 /*g) isolated 4 hr
after treatment with /3-propioIactone-3H as described in legend
for Chart 8. The chromatogram shown in Chart 8 was cut into
rectangles as described, eluted, and eluates counted as described
in Materials and Methods. The radioactivity
eluted in dpm/
rectangle is plotted as a function of position along the chromato
gram. Abbreviations as in Chart 8.
240O
2000-
SF2
Chart 8. Chromatogram of hydrolysate of RNA extracted from
tritiated /3-propiolactone (BPL-3H)-treated
mouse skin. Mice were
treated with 120 junóles (2070 #ic) of BPL-3H/mouse. Skins were
taken at 4 hr after BPL application. The extracted RNA was
hydrolyzed in IN HC1 (125 jig of RNA in 50 n\) at 100°Cfor
1 hour. The hydrolysate was chromatographed
in 2 dimensions
with unlabeled carrier 7-(2-carboxyethyl)guanine
(20 jug). Chromatograms of DNA hydrolysates were prepared and treated in
the same way. Shown are the positions of the origin, guanine
(G), 7-(2-carboxyethyl)guanine
(CEG), pyrimidine nucleotides
(PyNT), adenine (A), and solvent fronts for the first and second
dimensions (SFj and SF2).
648
ORIGIN
PyNT
Chart 10. Radioactive profile of paper chromatogram prepared
from hydrolysate of radioactive mouse skin DNA (150 /ig) iso
lated 4 hr after treatment with /3-propiolactone-3H as described
in legend for Chart 8. The DNA chromatogram was cut into
rectangles in the manner shown in Chart 8, the rectangles eluted,
and eluates counted as described in Materials and Methods. Ab
breviations as in Chart 8.
CANCER RESEARCH VOL. 28
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ß-Propiolactone Binding
of these 3 types of macromolecular binding bears a causal re
lation to initiation of tumorigenesis.
Characterization
of Nucleic
Acid
Binding
Products.
We
have previously reported that in vivo BPL-3H-treated DNA,
when hydrolyzed, chromatographed, and scanned for radio
activity, shows a single peak of radioactivity at the position
of 7-(2-carboxyethyl)guanine
(8). More recently we have
hydrolyzed and chromatographed BPL-3H-RNA from skin by
essentially the same procedure as was previously used for DNA.
The UV-absorbing spots on the chromatogram were located,
including fhat of carrier 7-(2-carboxyethyl)guanine.
The chro
matogram was cut into rectangles as indicated in Chart 8.
Each paper rectangle was cut into strips and eluted as de
scribed in Materials and Methods. Chart 9 shows the dpm
recovered from the chromatogram of the RNA hydrolysate as
a function of position along the chromatogram. One major
peak of radioactivity appeared exactly at the position of 7(2-carboxyethyl)guanine, with much smaller peaks in the re
gions of the pyrimidine nucleotides and adenine respectively.
Elution of a DNA chromatogram (Chart 10) showed one
major peak at the 7-(2-carboxyethyl)guanine
spot and much
smaller minor peaks in the regions of pyrimidine nucleotides
and adenine.
DISCUSSION
AND CONCLUSIONS
The finding that BPL-3H binds in vivo to protein as well as
to both nucleic acids adds BPL to the long list of carcinogens
for which the same observation has been made. A similar level
of carcinogen binding for all three macromolecules, as we have
found, has also been found for aromatic hydrocarbons in skin
(5) and for 2-acetylaminofluorene in liver (25), but not for
ethionine (14, 15) or dimethylaminoazobenzene (31) in liver.
The level of nucleic acid binding obtained with BPL, about 1
mmole BPL/mole DNA-P, is similar to that obtained by
Magee and Farber (24) for methylation of liver nucleic acids
by the carcinogen dimethylnitrosamine. This level is, however,
about 10 to 100 times that reported by other workers for
nucleic acid binding of carcinogenic aromatic amines, azo dyes,
and hydrocarbons (5, 25, 31, 37).
The high zero time binding for BPL is in contrast to the
observations for AAF (25) and aromatic hydrocarbons (17,
36), which must be metabolically activated before binding can
occur. The possibility that post-zero-time binding of BPL-3H
could be occurring in vitro was investigated, and it was con
cluded that in vitro binding to skin macromolecules occurred
only at zero time, not subsequently.
The binding of BPL-3H to skin DNA, RNA, and protein
reached maximum values at 2-12 hours after treatment. The
rate of decay of specific activity of BPL-3H DNA in vivo was
faster than either the rate of metabolic turnover or the rate
of in vitro depurination. This observation is similar to that for
dimethylnitrosamine-methylated
DNA. The DMN-methylated
DNA in liver shows an in vivo half-life for loss of 7-methyl
guanine of about 12 hours (9, 10). In contrast, methylated
DNA in vitro at pH 7.2 and 37°Cshows a half-life of about
200 hours (21). In the case of liver, in contrast to skin, DNA
turnover cannot account for the difference in in vivo and in
APRIL
1968
vitro rates, in view of the low mitotic index. Craddock and
Magee (10) suggest the possibility of enzymatic excision of
methylated guanines, by an enzyme which might be involved
in repair.4 Evidence for the operation of a repair mechanism
in mammalian cells has been found by Crathorn and Roberts
(11), who showed that the 35S label in HeLa cell DNA, in
troduced by treatment of cells with 3SS-labeled mustard gas,
was partially eliminated from DNA. It was found that the
specific radioactivity of 35S in DNA decreased more rapidly
than that of thymidine-3H introduced by prelabeling, and tht
apparent excision of mustard occurred during the first 17 hours
after treatment. Perhaps enzymatic excision may be contrib
uting to the rapid rate of decay of radioactivity in BPL-3HDNA. However, it seems likely that this rate could be com
pletely accounted for by the combined rates of metabolic
turnover and depurination.
The rate of decay of specific activity of BPL-3H-RNA in
vivo was faster than the rate of metabolic turnover. Since
depurination at neutral pH does not occur in polyribonucleotides (3), it seems likely that another mechanism was con
tributing to the rate of decay. Such a mechanism may involve
enzymatic excision and subsequent RNA chain scission. Alter
natively, the rate of decay of total RNA specific activity might
be completely accounted for by turnover of a specific RNA
species to which the BPL-3H is bound at a high level.
The rate of decay of specific activity of in vivo BPL-3Halkylated protein is about the same whether soluble, insoluble,
or total protein is determined. In addition, the level of BPL
binding is about the same for soluble and insoluble protein.
The observation that animals differing in susceptibility to
BPL show correlation of DNA, RNA, and protein binding, can
be contrasted with the observations for DAB (correlation of
ribosomal RNA binding, not DNA or protein binding) (31),
and AAF (correlation of RNA and protein binding, DNA cor
relation not reported) (25). Our data on the correlation of
DNA, RNA, and protein binding with tumorigenesis when dose
or mouse susceptibility are varied do not give suggestive evi
dence against the significance in tumorigenesis of any one of the
three types of macromolecular binding. Hence there remains
the possibility of a causal relationship between initiation of
tumorigenesis and BPL binding to DNA, RNA, or protein.
This question will be dealt with further in work to be reported
elsewhere.
With DNA and RNA, the level of binding by BPL-3H was
reduced to less than 1% of the 4-hour value by 3 weeks, while
protein binding was reduced to 10% of the 4-hour value at this
time. Initiation by BPL appears to be irreversible5; this de
mands that the compound produce some permanent change
in the informational content of the skin cells. Such a change
4 Warwick and Roberts' data on the persistent
binding of DAB
to liver DNA (40) suggest the absence of enzymatic repair in
liver. However, the possibility remains that there may be repair
enzymes which can recognize 7-methylguanine
but not the di
methylaminoazobenzene
binding product in DNA.
5 N. H. Colburn and R. K. Boutwell, unpublished results. After
initiation with 480 Amólesß-propiolactone/mouse, a similar tumor
incidence in papillomas/mouse
results whether promotion with
cortón oil is delayed one month or 5 months.
649
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N. H. Colburn and R. K. Boutwell
in the informational content must certainly involve a reaction
of the BPL with DNA, RNA, or protein, followed by some
critical event or events which irreversibly change the cell into
an initiated cell. Thus if the BPL is directly involved in a
change in the cell which is necessary in determining that its
descendants shall be neoplastic cells, that critical event must
occur before the bound form of the compound is eliminated,
or within a few weeks in this case. Perhaps this critical event
may involve DNA synthesis and cell division. It is of interest
in this context that BPL produces an increased rate of DNA
synthesis by 24 hr. Perhaps ensuing cell division allows for
selection of preneoplastic cells, similar to that suggested by
Prehn (29). Ford (16) has pointed out that, in normal somatic
tissues, selection is of a conservative nature; but in tissues
damaged by radiation or alkylating agents, selection is both
conservative and competitive, allowing the immediate cellular
survivors to express their different capacities to proliferate.
Fundamental to an understanding of carcinogenesis in molec
ular terms is the identification of the nature of the interaction
between the carcinogen and tissue receptors. Preliminary evi
dence of Muckerman and Boutwell (unpublished results) indi
cates that the products formed from in vivo BPL alkylation
of skin protein are S-carboxyethylcysteine and carboxyethylhistidine. Data reported here and previously (8) indicate that
7-(2-carboxyethyl)guanine
is the major product formed by in
vivo BPL alkylation of RNA or DNA. This formation of the
7-alkylguanine follows the pattern of all other monofunctional
alkylating agents reported.
The guanine-N-7 alkylation in DNA may exert a carcin
ogenic effect via depurination, or depurination and chain scis
sion, and thus produce frame shift mutagenesis or chromosome
deletion mutagenesis respectively, as discussed by Brookes and
Lawley (4). An example in which guanine-N-7 alkylation in
DNA is associated with mutagenesis in a mammalian system
has been reported by Swann (39), who finds that methyl
methanesulfonate, a mutagen which on injection into male rats
produces a dominant lethal mutation in sperm, also forms 7methylguanine in DNA and RNA of the testes. There is sug
gestive evidence that methyl methanesulfonate acts by pro
ducing chain breaks, since the chromosomes of treated rats
show deletions and translocations. It is of interest that several
esters of methanesulfonic acid are carcinogenic when applied
repeatedly to mouse skin (32).
An alternative mechanism by which BPL may be exerting
its carcinogenic effect is through mispairing of 7-(2-carboxyethyl)guanine in DNA with thymine or uracil during replica
tion or transcription, as discussed by Brookes and Lawley (4).
It may be noted here that the linear dose-response relationship
shown previously (8) for papillomas per mouse is consistent
with a single hit mechanism such as anomalous base pairing
and not with a mechanism involving chromosome deletions or
translocations; for the latter type of mechanism would give a
curve roughly proportional to the square of the dose, not a
straight line dose-response plot. Work concerned with the ques
tion of anomalous base pairing will appear elsewhere.
Borek and others (6, 38) have reported the occurrences of
methylated nucleic acids in several organisms. Another mech
anism by which guanine-N-7 alkylation might be envisaged to
650
exert a carcinogenic effect would be by disturbing the function
of naturally occurring methylated bases in nucleic acids. Al
though much remains to be learned about the function of these
abnormal bases, the available data suggest that they are neces
sary for normal specificity of recognition processes occurring
during gene transcription and translation. Revel and Littauer
(30) have reported that methyl-deficient phenylalanyl-transfer
RNA appears to make errors in its transfer function.6 One can
imagine a mechanism of carcinogenesis in which BPL alkyla
tion of transfer RNA disturbs the molecular configuration
brought about by the base methylation, and hence disturbs
the specificity of recognition in acceptor or transfer function.
It is of interest that Axel et cd. (1) have found that ethylated
soluble RNA from liver of ethionine-fed rats revealed one,
rather than the normal 3, leucyl-soluble RNA components on
methylated albumin-kieselguhr chromatography. Hence there
are a number of mechanisms whereby formation of 7-(2-carboxyethyOguanine in mouse skin nucleic acids may be signifi
cant in initiation of tumorigenesis by BPL. On the basis of the
work reported here, none of them can be eliminated.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. Carolyn Muckerman, Mrs. Kari
Haugli, Mrs. Dee Tuli, and Mrs. Julia Corbett for their excellent
technical assistance. We are grateful to Drs. Peter Brookes and
Loma Goshman for consultation in working out the modified
Kirby procedure used to extract nucleic acids and protein from
skin.
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651
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N. H. Colburn and R. K. Boutwell
^*m®*im
.**fc:Ã-fc^
Õ^S¿^7* *^v:;>^ *v
r
' •
!*%¿i. •••* *
-»
w••*•**-
**
-
-Vf"
'i '
_ *
fc'-Ã-
:.*,^Ã->i>
Fig. 1. Autoradiograph of mouse skin taken 2.5 hr after treatment
raphy was performed as described by Lesher (22). X 340.
652
•"
with 480 Amóles(500 fÃ-e)of tritiated
/8-propiolactone.
Autoradiog-
CANCER RESEARCH VOL. 28
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1968 American Association for Cancer Research.
The in Vivo Binding of β-Propiolactone to Mouse Skin DNA,
RNA, and Protein
N. H. Colburn and R. K. Boutwell
Cancer Res 1968;28:642-652.
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