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Intake of tritiated thymidine by epidermal cells of newborn mice treated with 7,12-dimethylbenz(a)anthracene
182
H. Garcia
The values of (Q&, for the various operating voltages normally used at present are given in the Table I, and the corresponding minimum thicknesses are also given considering Q -1.33 gm/cm3, the average density for various biological matter. The author expresses his thanks to Professor N. N. Das Gupta of Calcutta University for his interest and to Ministry of Scientific Research and Cultural Affairs, Government of India for financial assistance. TABLE
I. Xinimum
values for carbonaceous
mass thickness
Operating
u ( x 10 Is) cm2 (0 hnin
mWne
t min A
matter,
voltage (kV)
50
60
75
80
100
17.7 0.57 43.5
14.6 0.68 51.3
11.8 0.86 64.6
11.2 0.91 68.0
9.6 1.05 79.0
REFERENCES I. 2. 3. 4. 5. 6.
RURGE,
R. E. and SILVESTER, N. R., J. Uiophys. Rio&em. .J. H., Proc. Phys. Sot. (I,ondon) 69, 46 (1956). DE, hf. L., Xature 192, 547 (1961). HALL, C. E. and Ixou$, T., .I. iipp[. I-‘hys. 28, 1346 (1957). LEM, F., z. Naturjorsch. 9a, 185 (1954). SADH~KI
Cytol.
8, 1 (1960).
COUPLASD,
INTAKE
OF TRITIATED
THYMIDINE
OF NEWBORN
MICE
BY EPIDERMAL
TREATED
CELLS
WITH
7,12-DIMETHYLBENZ(a)ANTHRACENE’ H. GARCIA Division
of Oncology,
The Chicago
Medical Ill.,
Received
School, U.S.A.
February
Institute
of Medical
Research,
Chicago,
21, 1962*
PRELIMITS studies have demonstrated that the antimitotic action of the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) is not parallelled by a reduction in the uptake of tritiated thymidine by the epidermal cells of newborn mice. 1 This study was supported National Institutes of Health, * Revised version received Experimenfal
Cell Research
27
by grant CS-9212 U.S. Public Health April 10, 1962.
from the Service.
National
Cancer
Institute
of the
Tritiafed
ihymidine
by epidermal
183
cells of newborn mice
Swiss albino mice originally obtained from Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, and bred randomly in this laboratory were used. The animals are kept in plastic cages, with wood shavings, fed Rockland mouse diet and given water ad libitum. Twenty newborn animals of lessthan 15 hr of age, belonging to two different litters were selectedfor these studies, and divided, using five animals of each litter, into experimental groups of ten animals each. One group was painted TABLE
I.
Intake
of 3H-thymidine
Labeled
by epidermal with DMBA.
cells
cells of newborn
Grains
No. of cells labeled per thousand
so. of cells counted
DMIM Control
20,000 19,000
Mean
St. dev. ?A0
53.78 * 19 28.68 + 10
per cell No.
So. of cells counted
2.47 115
mice treated
of grai; per cell
St. dev.
Labeled mitoses %o
20 S_X.K 20.55 15.2
0.1 0.43
Mean
on the skin of the back with one drop of a solution of 0.5 per cent of DMBA (Eastman Organic Chemicals) in mineral oil (Superla 34, Standard Oil of Indiana). The other group was painted in the sameregion with one drop of mineral oil. After 24 hr the animals received 1 PC of tritiated thymidine (Schwarz Bioresearch Inc., Mount Vernon, New York) with a specific activity of 1.9 C/m&Zin 0.04 ml. of saline solution. The inoculation was given subcutaneously in the chest region. Two hours later the animals were sacrificed and the skin removed for autoradiographic studies [l, 2, 111. The skin was fixed in 10 per cent neutral formalin for 48 hr. Sections of 3 p were made, covered with stripping film (Kodak Ltd. ArlO) and exposed for 30 days. Before mounting permanently, they were stained with Ehrlich acid hematoxylin. One thousand cells of the basal cell layer of the epidermis were counted in each slide and the number of labeled cells recorded. Two different sections were counted from each animal. The results are given in Table I, which also includes the number of labeled mitoses. The number of labeled mitoses decreased24 hr after the application of the carcinogen. The occurrence of a decreasein the mitotic rate, following the application of polycyclic hydrocarbon carcinogens has been described. However, this is an early effect and is followed by a considerable increasein mitotic rate which persists [5, 6, 7, 12, 131. A decreasein mitotic rate may sometimes be related to a decreased DNA synthesis; this has been shown to be the case following the treatment with ionizing radiation and nitrogen mustard [3, 4, lo]. In the present experiment the decreasein the mitotic activity observed following the application of DMBA is not parallelled by a corresponding decrease in the number of cells synthesizing DNA, the mean number of labeled cells in the epidermis of newborn mice painted with DMBA is approximately double that of the control group. Since tritiated thymidine when inExperimental
Cell
Research
27
11. Garcia
184
Experimental
Cell Research
27
Trifiafed
fhymidine
by epidermal
cells of newborn mice
185
jetted into a living organism will label only those cells that are synthesizing DNA (81, it can be stated that in this case the number of cells in DNA synthesis is not decreased, and the probability that an increase had occurred is high. However, the level of synthesis in each individual cell is not increased as may be seen from the fact that the mean grain count per cell in both groups is similar. It is probable that the determination of mitotic alteration, in this case, involves factors other than those directly concerned with DNA synthesis [Cl]. It is impossible to say at the present time what additional functions may be altered by the carcinogen but it is clear that this may involve other unknown factors that trigger the onset of cell division. The author wishes to express his sincere gratitude to Dr. Philippe Shubik and Dr. Renato Baserga for their helpful indications and criticism and to Mr. Manuel Sueiro for his technical assistance. REFERENCES 1. 2. 3I 4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
BASERGA, R., KISIELESKI, \V. E. and HALVORST;N, K., Caruzr Ikeurch 20, 910 (1960). BASERGA, R. and NEMEROFF, K., Skin Technol. 37, 21 (1962). BODENSTEIS, D. and KONDRITZER, A. A ., .J. Exptf. Zoof. 107, 109 (1948). CATTANEO, S. -“1., QUASTLER, H. and SNERMAS, I:. G., RadiaLion Research 12, 587 (1960). DARIMERT, K., Acta Pathof. Alicrobiof. Scan!f. 53, 33 (1961). DAOUST, R., Actn Pathof. JlicroDiof. Stand. 45, 159 (1959). IVERSEN, S. and EDELSTEIS, .J. XI., Acta Pufhof. .Ilicrobiof. Scunrl. 30, 213 (1952). MACDONALD, R. A. and MALLORY, G. K., Lub. Inuesf. 8, 1547 (1959). MOSES, M. J. and TAYLOR, .J. I-i., Expff. Cell Research 9, 474 (1955). NEARY, G. J. and EVAXS, H. .J., in Proceedings of the Second United Nations International Conference on the Peaceful Uses of Atomic Energy, vol. 22, I3iological Effects of Radiations, p. 303. United Nations, Geneva, 1958. PELC, S. R., Intern. $7. Appf. Radiation and Isotopes 1, 172 (1956). RELLER, I-I. C. and COOFEH, %. K., Cancer Keseurch 4, 236 (1944). SCARPELLI, I>. G. and VON HAATX, E., Cancer Research 18, 657 (1958).
Stripping film dine. Ehrlich
autoradiographs acid hcmatoxylin,
Figs.
1 and
2.-Animals
Figs.
3 and
4.--Controls.
treated
of epidermal x 1200. with
cells
of newborn
mice
injected
with
tritiated
thymi-
DMBA.
Experiment&
Cell
Research
27
H. Garcia
The values of (Q&, for the various operating voltages normally used at present are given in the Table I, and the corresponding minimum thicknesses are also given considering Q -1.33 gm/cm3, the average density for various biological matter. The author expresses his thanks to Professor N. N. Das Gupta of Calcutta University for his interest and to Ministry of Scientific Research and Cultural Affairs, Government of India for financial assistance. TABLE
I. Xinimum
values for carbonaceous
mass thickness
Operating
u ( x 10 Is) cm2 (0 hnin
mWne
t min A
matter,
voltage (kV)
50
60
75
80
100
17.7 0.57 43.5
14.6 0.68 51.3
11.8 0.86 64.6
11.2 0.91 68.0
9.6 1.05 79.0
REFERENCES I. 2. 3. 4. 5. 6.
RURGE,
R. E. and SILVESTER, N. R., J. Uiophys. Rio&em. .J. H., Proc. Phys. Sot. (I,ondon) 69, 46 (1956). DE, hf. L., Xature 192, 547 (1961). HALL, C. E. and Ixou$, T., .I. iipp[. I-‘hys. 28, 1346 (1957). LEM, F., z. Naturjorsch. 9a, 185 (1954). SADH~KI
Cytol.
8, 1 (1960).
COUPLASD,
INTAKE
OF TRITIATED
THYMIDINE
OF NEWBORN
MICE
BY EPIDERMAL
TREATED
CELLS
WITH
7,12-DIMETHYLBENZ(a)ANTHRACENE’ H. GARCIA Division
of Oncology,
The Chicago
Medical Ill.,
Received
School, U.S.A.
February
Institute
of Medical
Research,
Chicago,
21, 1962*
PRELIMITS studies have demonstrated that the antimitotic action of the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) is not parallelled by a reduction in the uptake of tritiated thymidine by the epidermal cells of newborn mice. 1 This study was supported National Institutes of Health, * Revised version received Experimenfal
Cell Research
27
by grant CS-9212 U.S. Public Health April 10, 1962.
from the Service.
National
Cancer
Institute
of the
Tritiafed
ihymidine
by epidermal
183
cells of newborn mice
Swiss albino mice originally obtained from Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, and bred randomly in this laboratory were used. The animals are kept in plastic cages, with wood shavings, fed Rockland mouse diet and given water ad libitum. Twenty newborn animals of lessthan 15 hr of age, belonging to two different litters were selectedfor these studies, and divided, using five animals of each litter, into experimental groups of ten animals each. One group was painted TABLE
I.
Intake
of 3H-thymidine
Labeled
by epidermal with DMBA.
cells
cells of newborn
Grains
No. of cells labeled per thousand
so. of cells counted
DMIM Control
20,000 19,000
Mean
St. dev. ?A0
53.78 * 19 28.68 + 10
per cell No.
So. of cells counted
2.47 115
mice treated
of grai; per cell
St. dev.
Labeled mitoses %o
20 S_X.K 20.55 15.2
0.1 0.43
Mean
on the skin of the back with one drop of a solution of 0.5 per cent of DMBA (Eastman Organic Chemicals) in mineral oil (Superla 34, Standard Oil of Indiana). The other group was painted in the sameregion with one drop of mineral oil. After 24 hr the animals received 1 PC of tritiated thymidine (Schwarz Bioresearch Inc., Mount Vernon, New York) with a specific activity of 1.9 C/m&Zin 0.04 ml. of saline solution. The inoculation was given subcutaneously in the chest region. Two hours later the animals were sacrificed and the skin removed for autoradiographic studies [l, 2, 111. The skin was fixed in 10 per cent neutral formalin for 48 hr. Sections of 3 p were made, covered with stripping film (Kodak Ltd. ArlO) and exposed for 30 days. Before mounting permanently, they were stained with Ehrlich acid hematoxylin. One thousand cells of the basal cell layer of the epidermis were counted in each slide and the number of labeled cells recorded. Two different sections were counted from each animal. The results are given in Table I, which also includes the number of labeled mitoses. The number of labeled mitoses decreased24 hr after the application of the carcinogen. The occurrence of a decreasein the mitotic rate, following the application of polycyclic hydrocarbon carcinogens has been described. However, this is an early effect and is followed by a considerable increasein mitotic rate which persists [5, 6, 7, 12, 131. A decreasein mitotic rate may sometimes be related to a decreased DNA synthesis; this has been shown to be the case following the treatment with ionizing radiation and nitrogen mustard [3, 4, lo]. In the present experiment the decreasein the mitotic activity observed following the application of DMBA is not parallelled by a corresponding decrease in the number of cells synthesizing DNA, the mean number of labeled cells in the epidermis of newborn mice painted with DMBA is approximately double that of the control group. Since tritiated thymidine when inExperimental
Cell
Research
27
11. Garcia
184
Experimental
Cell Research
27
Trifiafed
fhymidine
by epidermal
cells of newborn mice
185
jetted into a living organism will label only those cells that are synthesizing DNA (81, it can be stated that in this case the number of cells in DNA synthesis is not decreased, and the probability that an increase had occurred is high. However, the level of synthesis in each individual cell is not increased as may be seen from the fact that the mean grain count per cell in both groups is similar. It is probable that the determination of mitotic alteration, in this case, involves factors other than those directly concerned with DNA synthesis [Cl]. It is impossible to say at the present time what additional functions may be altered by the carcinogen but it is clear that this may involve other unknown factors that trigger the onset of cell division. The author wishes to express his sincere gratitude to Dr. Philippe Shubik and Dr. Renato Baserga for their helpful indications and criticism and to Mr. Manuel Sueiro for his technical assistance. REFERENCES 1. 2. 3I 4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
BASERGA, R., KISIELESKI, \V. E. and HALVORST;N, K., Caruzr Ikeurch 20, 910 (1960). BASERGA, R. and NEMEROFF, K., Skin Technol. 37, 21 (1962). BODENSTEIS, D. and KONDRITZER, A. A ., .J. Exptf. Zoof. 107, 109 (1948). CATTANEO, S. -“1., QUASTLER, H. and SNERMAS, I:. G., RadiaLion Research 12, 587 (1960). DARIMERT, K., Acta Pathof. Alicrobiof. Scan!f. 53, 33 (1961). DAOUST, R., Actn Pathof. JlicroDiof. Stand. 45, 159 (1959). IVERSEN, S. and EDELSTEIS, .J. XI., Acta Pufhof. .Ilicrobiof. Scunrl. 30, 213 (1952). MACDONALD, R. A. and MALLORY, G. K., Lub. Inuesf. 8, 1547 (1959). MOSES, M. J. and TAYLOR, .J. I-i., Expff. Cell Research 9, 474 (1955). NEARY, G. J. and EVAXS, H. .J., in Proceedings of the Second United Nations International Conference on the Peaceful Uses of Atomic Energy, vol. 22, I3iological Effects of Radiations, p. 303. United Nations, Geneva, 1958. PELC, S. R., Intern. $7. Appf. Radiation and Isotopes 1, 172 (1956). RELLER, I-I. C. and COOFEH, %. K., Cancer Keseurch 4, 236 (1944). SCARPELLI, I>. G. and VON HAATX, E., Cancer Research 18, 657 (1958).
Stripping film dine. Ehrlich
autoradiographs acid hcmatoxylin,
Figs.
1 and
2.-Animals
Figs.
3 and
4.--Controls.
treated
of epidermal x 1200. with
cells
of newborn
mice
injected
with
tritiated
thymi-
DMBA.
Experiment&
Cell
Research
27