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The Effect of Dimethyl Sulfoxide on the Severity of Radiation Skin Lesions*
Vol. 52, No. 3
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1969 by The Williams & Wilkins Co.
THE EFFECT OF DIMETHYL SULFOXIDE ON THE SEVERITY OF RADIATION SKIN LESIONS* RONALD F. HAGEMANN, Pn.D.t, LARRY L. SCHENKEN, MA. AND TITUS C. EVANS, PH.D.
Local protection of the skin from radiation damage, by topical chemical application, is of considerable interest from both a radiobiologi-
50%, or 20% DMSO; 50% DMSO plus 50%
topical skin radioprotector. First, it has been shown to be a radioprotector on both whole animal (2) and cellular (3) levels. Second, it passes through intact skin with relative ease (4). Third, it can act as a percutaneous penetrant carrier for a number of organic materials (5). For this latter reason, in addition to using
quamation; 2—3, dry desquamation; 3—5, moist desquamation—< 50% skin loss; 5—6, >50% skin loss.
1:1000 epinephrine; or water) was applied topically to the area to be exposed on one thigh, and water was applied to the other thigh. The number cal and radiotherapeutical point of view. A of animals in each group varied from 4 to 10. The number of substances, e.g. mereaptoethylaniine upper thighs of each animal were irradiated (MEA), have been found which are effective through two 2.1 cm square ports in a 0.64 cm skin radioprotectors when administered by in- thick lead sheet, which shielded the remainder of the animal. The two thighs of each animal were travenous or subcutaneous injection (see Ref- irradiated concomitantly. Physical factors of irerence 1 for review). Attempts to modify radia- radiation were 250 kVp, 30 mA, 1 mm Al filtration, tion skin damage by topical treatment have not HVL 026 mm Cu, target to skin distance 25 cm, been very successful. Unfortunately, intrave- exposure rate 1230 R/minute as measured in air at the level of the skin by a Victoreen condenser nous injection affords systemic protection, and R-meter. The total exposure was 6500 R. subcutaneous infiltration is tedious and may The animals were examined periodically after exposure. Two investigators scored the skin lesions also offer some generalized protection. Several properties of dimethyl sulf oxide according to the following general scheme: 0—1, (DMSO) suggested its possible usefulness as a erythema; 1—2, severe erythema—mild dry desRESULTS AND DISCUSSION
The data obtained are shown in Figure 1. It is apparent that DMSO, an established radioprotector in other systems, riot only did not protect against radiation skin damage under DMSO alone, experiments were performed with these experimental conditions, but in high conDMSO plus MEA (a very effective skin radio- centration resulted in marked skin radiosensiprotector), and with DMSO plus epinephrine tization (Figs. 1 and 2). Furthermore, this sen(in an attempt to decrease local oxygen tension sitizing action of 100% DMSO was not negated and hence afford protection). It has not been by the addition of MEA. It should be pointed established however, that DMSO can act as a out, that the 20 minute application of MEA penetrant carrier for either MEA or epineph- may not have been of sufficient duration to observe a protective effect. Statistical analysis rine.
of the data in Figure 1 (t test) showed a
MATERIALS AND METHODS
highly significant difference (largest p < .001)
Male rats (Simonsen) weighing 400—500 gm were
between the 100% DMSO, or 100% DMSO
used. The animals were numbered and the hair on
the thigh and lower back was clipped. Twenty plus MEA, and water groups. No apparent skin minutes prior to irradiation the test solution (100% damage was observed after topical application DMSO; 100% DMSO plus MBA, 60 mg/ml; 80%, of 100% DM50 or 100% DMSO plus MEA to Received July 1, 1968; accepted for publication unirradiated animals, e.g. Figure 2, F. Lower August 30, 1968. concentrations of DMSO, or the addition of This study was supported by grants from the American Cancer Society, and the Biology Di- epinephrine, did not alter the progression of radiation skin damage from that observed in vision, U.S.A.E.C. * From the RadIation Research Laboratory, the water controls. College of Medicine, University of Iowa, Iowa It should be noted that the data in Figure 1 City, Iowa 52240. f Present address: Division of Radiation Bi- describe the onset and progression of the radiaology. Department of Radiology, Allegheny Gen- tion damage. The repair phase was exceedingly eral Hospital, 320 E. North Avenue, Pittsburgh, difficult to categorize according to the scheme Pennsylvania 15212. 277
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
278
employed and hence no attempt was made to apparent difference between the 100% DM50 quantitate this aspect. However, by about 25 or 100% DSMO plus MEA and water groups. days after irradiation, there was no longer any At the exposure level employed, all skin lesions eventually healed (usually within 2—3 months 6
C
.1
5
after exposure). We have repeated these experiments using a lower exposure (5000 H) and the results qual-
itatively confirm those reported here. Again, radiosensitization was found with high concentrations of DM80.
4
It is likely that the skin radiosensitization caused by 100% DM80 is pharmacological in
nature. Bradham and Sample (6) have re-
>-3
ported increased skin temperature after topical
application of high concentrations (70%) of DMSO. This is probably due to the heat of
a U2 0
6500 R
W W
.—. WATER
(0
100% DM50 IOO%DMSO+MEA 60% DM50
WI 0
• 50%DMSO * 50% DM50 +EPI. o 20%DMSO 6 4 12 io TIME AFTER EXPOSURE, days
i
o
hydration resulting from the reaction of DMSO with tissue water. In separate experiments, we have noticed marked accumulation of injected
Evans' blue in skin areas treated with 100% DMS0 (unpublished observations). This regional hyperemia caused by DMSO in high
concentrations may explain its radiosensitizing action. The increased blood flow to the treated FIG. 1. Degree of injury (see text) vs days alter area would raise the local oxygen tension. Thus X-irradiation. For clarity, curves are shown for water and 100% DM80 only. EPI = epinephrine. those cells, which may have been hypoxic (and
lift i
L?,
Fio. 2. Photograph of typical skin lesions taken 8 days after X-irradiation with 6500 R. Pretreatment was with either 100% DM80 or water. A. DMSO left (L), water right (H), B. DM80 (II), water (L). C. DM80 (L), water (R). D. DM80 (L), water (R). E. DM80 (R), water (L). F. DM80 (L and R), unirradiated.
DMSO AND RADIATION SKIN LESIONS
hence relatively radioresistant) prior to treat-
279
hypoxic cells, thereby increasing radiosensitiv-
ment would have been irradiated in a more ity. oxygenated (radiosensitive) state. This inter-
pretation seems consistent with the well-known "oxygen effect" in radiobiology, and is further supported by the finding of van den Brenk (7)
REFERENCES 1. Bacq, Z. M.: Chemical Protection against Ionizing Radiation. Charles C Thomas, Springfield, Illinois, 1965.
M. J.: The radioprotective acthat rat skin radiosensitivity is increased in 2. Ashwood-Smith, tion of dimethyl suiphoxide and various other
animals breathing 100% oxygen in place of air. SUMMARY
sulphoxides. mt. J. Radiat. Biol., 3: 41, 1961. 3. Vos, 0., Kaalin, A. C. and Budke, L.: Radiation protection by a number of substances prevent-
ing freezing damage. I. protection of mammalian cells in vitro. mt. J. Radiat. Biol., 9: Under the experimental conditions employed, 133, 1965. topical application of various concentrations of 4. Kligman, A. M.: Topical pharmacology and dimethyl sulfoxide did not result in skin radiotoxicology of dimethyl sulf oxide—Part I. J. A. M. A., 193: 796, 1965. protection. Combinations of DMSO and MEA 5. Stoughton, R. B. and Fritsch, W.: Influence of or epinephrine were equally ineffective. DMSO, dimethyl sulfoxide (DMSO) on human peran established radioprotective agent, in high cutaneous absorption. Arch. Derm., 90: 512,
1964. concentration, resulted in marked skin radio6. Bradham, G. B. and Sample, J. J.: The vascular sensitization. This latter finding may be exand thermal effects of dimethyl sulfoxide. Ann. N. Y. Acad. Sci., 141: 225, 1967. plained on the basis of the local hyperemie 7. van den Brenk, H. A. S.: Increased radiosensiresponse to the hydration of DMSO, resulting tivity of skin and normal tissues in high pres.. in an increased oxygen tension in previously sure oxygen. Amer. J. Roent., 97: 1007, 1966.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1969 by The Williams & Wilkins Co.
THE EFFECT OF DIMETHYL SULFOXIDE ON THE SEVERITY OF RADIATION SKIN LESIONS* RONALD F. HAGEMANN, Pn.D.t, LARRY L. SCHENKEN, MA. AND TITUS C. EVANS, PH.D.
Local protection of the skin from radiation damage, by topical chemical application, is of considerable interest from both a radiobiologi-
50%, or 20% DMSO; 50% DMSO plus 50%
topical skin radioprotector. First, it has been shown to be a radioprotector on both whole animal (2) and cellular (3) levels. Second, it passes through intact skin with relative ease (4). Third, it can act as a percutaneous penetrant carrier for a number of organic materials (5). For this latter reason, in addition to using
quamation; 2—3, dry desquamation; 3—5, moist desquamation—< 50% skin loss; 5—6, >50% skin loss.
1:1000 epinephrine; or water) was applied topically to the area to be exposed on one thigh, and water was applied to the other thigh. The number cal and radiotherapeutical point of view. A of animals in each group varied from 4 to 10. The number of substances, e.g. mereaptoethylaniine upper thighs of each animal were irradiated (MEA), have been found which are effective through two 2.1 cm square ports in a 0.64 cm skin radioprotectors when administered by in- thick lead sheet, which shielded the remainder of the animal. The two thighs of each animal were travenous or subcutaneous injection (see Ref- irradiated concomitantly. Physical factors of irerence 1 for review). Attempts to modify radia- radiation were 250 kVp, 30 mA, 1 mm Al filtration, tion skin damage by topical treatment have not HVL 026 mm Cu, target to skin distance 25 cm, been very successful. Unfortunately, intrave- exposure rate 1230 R/minute as measured in air at the level of the skin by a Victoreen condenser nous injection affords systemic protection, and R-meter. The total exposure was 6500 R. subcutaneous infiltration is tedious and may The animals were examined periodically after exposure. Two investigators scored the skin lesions also offer some generalized protection. Several properties of dimethyl sulf oxide according to the following general scheme: 0—1, (DMSO) suggested its possible usefulness as a erythema; 1—2, severe erythema—mild dry desRESULTS AND DISCUSSION
The data obtained are shown in Figure 1. It is apparent that DMSO, an established radioprotector in other systems, riot only did not protect against radiation skin damage under DMSO alone, experiments were performed with these experimental conditions, but in high conDMSO plus MEA (a very effective skin radio- centration resulted in marked skin radiosensiprotector), and with DMSO plus epinephrine tization (Figs. 1 and 2). Furthermore, this sen(in an attempt to decrease local oxygen tension sitizing action of 100% DMSO was not negated and hence afford protection). It has not been by the addition of MEA. It should be pointed established however, that DMSO can act as a out, that the 20 minute application of MEA penetrant carrier for either MEA or epineph- may not have been of sufficient duration to observe a protective effect. Statistical analysis rine.
of the data in Figure 1 (t test) showed a
MATERIALS AND METHODS
highly significant difference (largest p < .001)
Male rats (Simonsen) weighing 400—500 gm were
between the 100% DMSO, or 100% DMSO
used. The animals were numbered and the hair on
the thigh and lower back was clipped. Twenty plus MEA, and water groups. No apparent skin minutes prior to irradiation the test solution (100% damage was observed after topical application DMSO; 100% DMSO plus MBA, 60 mg/ml; 80%, of 100% DM50 or 100% DMSO plus MEA to Received July 1, 1968; accepted for publication unirradiated animals, e.g. Figure 2, F. Lower August 30, 1968. concentrations of DMSO, or the addition of This study was supported by grants from the American Cancer Society, and the Biology Di- epinephrine, did not alter the progression of radiation skin damage from that observed in vision, U.S.A.E.C. * From the RadIation Research Laboratory, the water controls. College of Medicine, University of Iowa, Iowa It should be noted that the data in Figure 1 City, Iowa 52240. f Present address: Division of Radiation Bi- describe the onset and progression of the radiaology. Department of Radiology, Allegheny Gen- tion damage. The repair phase was exceedingly eral Hospital, 320 E. North Avenue, Pittsburgh, difficult to categorize according to the scheme Pennsylvania 15212. 277
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
278
employed and hence no attempt was made to apparent difference between the 100% DM50 quantitate this aspect. However, by about 25 or 100% DSMO plus MEA and water groups. days after irradiation, there was no longer any At the exposure level employed, all skin lesions eventually healed (usually within 2—3 months 6
C
.1
5
after exposure). We have repeated these experiments using a lower exposure (5000 H) and the results qual-
itatively confirm those reported here. Again, radiosensitization was found with high concentrations of DM80.
4
It is likely that the skin radiosensitization caused by 100% DM80 is pharmacological in
nature. Bradham and Sample (6) have re-
>-3
ported increased skin temperature after topical
application of high concentrations (70%) of DMSO. This is probably due to the heat of
a U2 0
6500 R
W W
.—. WATER
(0
100% DM50 IOO%DMSO+MEA 60% DM50
WI 0
• 50%DMSO * 50% DM50 +EPI. o 20%DMSO 6 4 12 io TIME AFTER EXPOSURE, days
i
o
hydration resulting from the reaction of DMSO with tissue water. In separate experiments, we have noticed marked accumulation of injected
Evans' blue in skin areas treated with 100% DMS0 (unpublished observations). This regional hyperemia caused by DMSO in high
concentrations may explain its radiosensitizing action. The increased blood flow to the treated FIG. 1. Degree of injury (see text) vs days alter area would raise the local oxygen tension. Thus X-irradiation. For clarity, curves are shown for water and 100% DM80 only. EPI = epinephrine. those cells, which may have been hypoxic (and
lift i
L?,
Fio. 2. Photograph of typical skin lesions taken 8 days after X-irradiation with 6500 R. Pretreatment was with either 100% DM80 or water. A. DMSO left (L), water right (H), B. DM80 (II), water (L). C. DM80 (L), water (R). D. DM80 (L), water (R). E. DM80 (R), water (L). F. DM80 (L and R), unirradiated.
DMSO AND RADIATION SKIN LESIONS
hence relatively radioresistant) prior to treat-
279
hypoxic cells, thereby increasing radiosensitiv-
ment would have been irradiated in a more ity. oxygenated (radiosensitive) state. This inter-
pretation seems consistent with the well-known "oxygen effect" in radiobiology, and is further supported by the finding of van den Brenk (7)
REFERENCES 1. Bacq, Z. M.: Chemical Protection against Ionizing Radiation. Charles C Thomas, Springfield, Illinois, 1965.
M. J.: The radioprotective acthat rat skin radiosensitivity is increased in 2. Ashwood-Smith, tion of dimethyl suiphoxide and various other
animals breathing 100% oxygen in place of air. SUMMARY
sulphoxides. mt. J. Radiat. Biol., 3: 41, 1961. 3. Vos, 0., Kaalin, A. C. and Budke, L.: Radiation protection by a number of substances prevent-
ing freezing damage. I. protection of mammalian cells in vitro. mt. J. Radiat. Biol., 9: Under the experimental conditions employed, 133, 1965. topical application of various concentrations of 4. Kligman, A. M.: Topical pharmacology and dimethyl sulfoxide did not result in skin radiotoxicology of dimethyl sulf oxide—Part I. J. A. M. A., 193: 796, 1965. protection. Combinations of DMSO and MEA 5. Stoughton, R. B. and Fritsch, W.: Influence of or epinephrine were equally ineffective. DMSO, dimethyl sulfoxide (DMSO) on human peran established radioprotective agent, in high cutaneous absorption. Arch. Derm., 90: 512,
1964. concentration, resulted in marked skin radio6. Bradham, G. B. and Sample, J. J.: The vascular sensitization. This latter finding may be exand thermal effects of dimethyl sulfoxide. Ann. N. Y. Acad. Sci., 141: 225, 1967. plained on the basis of the local hyperemie 7. van den Brenk, H. A. S.: Increased radiosensiresponse to the hydration of DMSO, resulting tivity of skin and normal tissues in high pres.. in an increased oxygen tension in previously sure oxygen. Amer. J. Roent., 97: 1007, 1966.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.