ERUPTIONS OF CORIAL MELANOPHORES ANDGENERAL CUTANEOUS MELANOSIS IN THE

GOLDFISH (CARASSIUS AURATUS)FOLLOWING EXPOSURE

TO X-RAY

GEORGE MILTON Sl\UTH, M.D.'

(From the Anatomical Laboratory, School of Medicine, Yale Uuioereitu, NewHallen, Connecticut)

The subject of cutaneous pigment and of melanophores in manand in the lower animals has been widely studied in recent years.Discussions of some of the many problems connected with thissubject appear in the works of Bloch (1), Biedermann (2), Miescher(3), Masson (4), Mierowsky (5), Ewing (6), Becker (7), Peck (8),Foot (9), and Cordier (10). It is recognized that the unravellingof the general problems of melanophores is definitely related to thesubject of the pigmented tumors.

Bloch (1), in particular, has stressed the view that melanin isproduced in the skin by melanoblasts as a result of enzyme action.In man, cutaneous melanoblasts are found in the epidermis and inthe corium (Bloch, 1; Becker, 7; Peck, 8). The melanoblastic cellsof the epidermis are principally basal cells, and certain other cellswith dendrites, limited to the epidermis. In the human corium,melanoblasts exist, according to Bloch (1), in the collection ofpigmented cells in the sacral region known as the Mongolian spot,and also in the blue colored nevi of the skin. They occur in thecorium of the monkey (Toldt, 11). They are found in the Japanesesilk fowl (Kuklenski, 12), and in the grey mouse (Steiner-Wourlisch,13). The corial melanophores found in man and in certain warm­blooded animals are thought to be related to the corial melano­phores seen in such abundance in cold-blooded animals.

In fishes, Ballowitz (14, 15) was the first to demonstrate thehistological connection between melanophores of the corium andthe peripheral nervous system. The well known contractions andexpansions of these pigmented cells in fishes are determined byimpulses from centers in the medulla and cord, by means ofpigment motor nerve fibers, and by the sympathetic nerves, asdemonstrated by von Frisch (16, 17). The earlier work of Soldan

'Aided hy the Blossom Fund, Yale University.

863

864 GEORGE MILTON SMITH

(18) and the more recent studies of Masson (4) have established theorigin of the nevus cell in man from the nervous system. In thisconnection Ewing (6) states that" present indications, therefore,point to the conclusion that nevus cells and chromatophores aredifferent phases of the same cell order, and that both are derivedfrom the nervous system and are free from any epithelial en­tanglements."

Whereas melanophores in fishes have been studied by manyinvestigators, the experimental production of melanosis has re­ceived but little attention. From the standpoint of melanosis infishes, the recent studies of Reed and Gordon (19) showed that inhybrids of the Mexican killifish an increase in melanophoresexisted to form actually melanotic tumor masses. Experimentalobservations of Fukui (20) are referred to in the discussion, below.

In a previous paper (Smith, 21), it was noted that in certainexperimental wounds of the goldfish (Carassius auratus) aneruption of melanophores varying in intensity in different fishesoccurred in the region of the wound and frequently at remotecutaneous points. These melanophores were regarded as ofimportance in the mechanism of tissue repair and defense. Theeffects of a number of conditions, such as temperature, darkness,and repeated trauma, upon the production of melanophoreselicited by mechanical injury were studied.

It seemed of interest to learn if exposure of goldfishes to x-rayswould likewise cause an eruption of melanophores. This proved tobe the case. Through the generous co-operation of Dr. WilliamA. LaField and Mr. E. E. Furbursh of Yale University X-RayDepartment, a small series of experiments was undertaken on 47goldfishes, the results of which are here reported.

METHOD

Small goldfishes, measuring 4.5 to 10 em. in length fromsnout to base of tail, and preferably of deep reddish tinge, wereselected for the experiments. Before x-raying, an examination ofall cutaneous regions was made with a dissecting microscope toinsure absence of visible corial melanophores, or to chart theirexact location if present.

During exposure to the x-rays it was found advisable toanesthetize the fishes by placing them in a solution of chloretone 1part, water 2000 parts. This insured a quiet state and eliminateddanger of injury from excessive struggling. The solution ofchloretone used for this purpose is harmless and does not evoke aneruption of melanophores.

ERUPTIONS OF MELANOPHORES IN GOLDFISH 865

The anesthetized fishes were placed on the right side on alinen sheet folded into several thicknesses, the left side of thebody being thus exposed directly to the x-ray tube. A small pieceof cotton, moistened in tap water, was placed over the exposedoperculum of each fish to keep the region of the gills slightlymoistened during the period of exposure.

A unit of dosage sufficient to produce an erythema of thehuman skin was used as a convenient measure of x-ray dosage.One unit of erythema dose consisted of 100 k. v. p., 5 milliamperes,8 inch target, skin distance, no filter, 72 second exposure. Theapparatus had a 250 k. v. capacity.

RESULTS OF EXPERIMENTS

In order to show the results of experiments the following tablewas arranged.

TABLE 1

Number of EarliestX-Ray Number of Site Exposed Reaction Poaitive Day of

E~hema Fishes or Negative Melanophorenits Eruption

-- ---I 3 Tail Negative 0

----2 3 Tail Negative 0

3 3 Entire body Negative 0

4 3 Tail and one third of body PositiveSlight 5

---5 3 Entire body Positive

Slight 5

(j 4 Tail and one third of body PositiveSlight 5

7 10 Entire body PositiveMarked 6

8 3 Entire body PositiveMarked 6

---9 6 Entire body Positive

Marked 5----

10 9 Entire body PositiveMarked I 6

NOTE: One unit of erythema dose = 100 k. p. v., 5 milliamperes, 8 inch target, skindistance, no filter, 72 second exposure. Forty-seven fishes kept under diffuse laboratorydaylight conditions in tanks of still water, supplied by a current of air. Temperature ofwater 70° F.

866 GEORGE MILTON SMITH

Disregarding occasional very small groups of melanophoreswhich form in the tip of the caudal fin from trauma due to strug­gling, and considering a positive reaction one in which numerousmelanophores appeared in the different exposed surfaces, it will beseen from the foregoing table that the more striking eruptions ofmelanophores occurred after the larger doses of from 7 to 10 units,at about the fifth or sixth day. The extent of the eruption wassubject to variations in intensity for different fishes.

PUOTOMIClWGHAI'1l 1. MEI.ANOPHORES ON A BODY SCAI.~; T.<\.KEN ~'ROM FISH

Fh;EN IN PLAT~; 1, FIGs. 1 AND 2, ON TUE 18TH DAY AFTER A 7-l'NIT EXPOSUUE OF

X-RAY

Depigmentation has begun, and a good deal of pigment detritus appears scatteredamong normal and degenerating cells. X 125.

A typical reaction in a fish measuring 7 em. is seen in Plate I,Fig. 1, sixteen days after x-ray exposure of seven units. Thepigmentation occupied chiefly the left side or exposed side of thebody. It occurred to some extent on all the fins. The tissues onthe right side preserved their normal coloring. In this fish theeruption began on the 6th day as a few microscopically visiblemelanophores along the side of the body above the lateral linecanal and on the left side of the head. Evidence of degenerationof melanophores was noted on the 18th day (Photomicrograph 1)and complete depigmentation was accomplished by the end oftwenty-nine days. Plate I, Fig. 2 shows this fish with complete

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ERUPTIONS OF MELANOPHORES IN GOLDFISH 867

disappearance of black pigment and restoration to normal color onthe 29th day after exposure.

An instance of intense general cutaneous melanosis photo­graphed sixteen days after 7 units of x-ray is seen in Plate I, Fig. 3.A normal goldfish was photographed at the same time to show thecontrast between normal coloring and pigmentation from x-ray.Melanophores composing this eruption offered the widest possiblerange in variety of shape, intensity of pigmentation, states of

PHOTOMWROGRAPH 2. FORMS OF MELANOPHORES SEEN ON A BODY SCALE RE­MOVED t'ROM THt; FI~H SHOWN IN PI,ATE 1, FIGS. 3 AND 4, EIGHTEEN DAYS AF'l'Elt A

7-UNIT EXPOSURE OF X-RAY. X 125

contraction or expansion, and the arrangement of the interlacingprocesses to form massed pigmented areas. Photomicrograph 2shows several of these cells on a body scale removed from thisfish at the height of eruption, eighteen days. Plate I, Fig. 4,shows the same fish, in a state of depigmentation, recovering fromgeneral cutaneous melanosis, photographed on the 31st day afterx-ray exposure. Complete recovery occurred in three months.

DISCUSSION

Fukui (20), who has made a study of melanophores and pigmentpatterns of the goldfish during stages of growth, found that in thenewly hatched fish melanophores are arranged at first along certaindefinite lines of the body. When the fish reaches a length of

868 GEORGE MILTON SMITH

about 1 cm., melanophores greatly increase in number so that thefish turns olive brown. At a length of 2 em. melanophores and alsoxanthophores are destroyed and depigmentation begins. De­pigmentation is not constant, may stop in mid-course, or maynever even be initiated. As a result of depigmentation the variousfinal colors and patterns of the goldfish are produced.

Fukui (20) found a new formation of melanophores afterinjections of adrenalin, and when fishes were kept in solutions ofchemicals such as sodium and calcium chloride. In experiments ofregeneration of the caudal fin in the black-backed goldfish, new tis­sues were developed producing melanophores, and it was suggestedthat this is due to tyrosinase in the skin, which would form melanin.

The possible functions of melanophores in fishes have beendiscussed in a previous paper (Smith, 21), in which was described,also, the occurrence of pigmentation in goldfishes by the productionof melanophores through mechanical injury, with the subsequentdegeneration of these pigment cells.

In the present experiments on goldfishes, x-ray exposure hasbeen found to call forth a visible eruption of melanophores, similarto that following mechanical trauma. Both forms of eruptionshowed a similar process of degeneration of melanophores with aclearing up of pigmented areas formed in the experiment.

The exact significance of these experimentally evoked melano­phores, other than that they appear in relation to repair anddefense of tissue, is for the moment only a matter of conjecture.A microscopic study of paraffin sections, prior to and at the time oferuption, gives the impression that x-ray-elicited melanophoreshave their source in delicate, narrow, irregular corial connective­tissue cells which exist normally either without pigment granules oras cells with a small amount of extremely fine, dust-like, brownishpigment as seen under high magnification. Chemical changes inthe corium as the result of x-ray exposure doubtless cause anincreased production of pigment participated in by a larger numberof cells than normal. Such melanophores, much enlarged, filledwith a great amount of pigment, soon become visible as they ar­range themselves directly under the transparent epithelium of thefish as the typical dendritic black cell with its countless smallnearly uniform granules of pigment. Great numbers of heavilypigmented melanophores, massed together with interlacing proc­esses, form dense pigmented patches visible to the eye. Whenpigmentation is far reaching, a general cutaneous melanosis occurs,

ERUPTIONS OF MELANOPHORES IN GOLDFISH 869

the entire surface of the fish appears black, and melanophores maybe seen as well on the mesial side of the operculum, in the buccalcavity, nasal depression, anal mucous membrane, and in the periph­eryof the cornea; sometimes a few cells are seen, also, in the gills.

Miescher (22) states that skin reactions in man from x-rayappear often clinically in waves of cutaneous redness or erythemaduring the first, third, and sixth week after -exposure. He findsthat pigmentation of the basal cells of the epidermis may beassociated with any of the waves of redness and may last as long astwo years. In the goldfish, depigmentation, with complete disap­pearance of pigmented areas by a process of melanophore degener­ation, is for the most part quickly accomplished, taking from twoweeks to a little over a month in tanks of 70° F. Where pig­mentation remained extensive, particularly when death resulted infishes while in an extreme state of melanosis (seen in 4 instances),it was thought not unlikely that some other factor, such as asecondary invasion of damaged skin by bacteria and fungi,complicated the inflammation evoked by x-rays.

It is of interest to note the relatively great resistance to x-rayexposure displayed by the goldfish. A single dose of ten times thehuman erythema dose could be withstood by some fishes, withoutthe development of burns or sloughing of tissues.

SUMMARY

By means of x-rays, eruptions of melanophores have beenproduced in the goldfish. The production of melanophores maybe so great as to cause a general cutaneous melanosis. De­pigmentation occurs usually in the course of several weeks.General cutaneous melanosis, aided by secondary infections of theskin, may result in death of the fish. The goldfish appears rela­tively resistant to x-ray exposure.

REFERENCES

1. BLOCH, B.: Das Pigment, in Handbuch d. Haut- u. Geschlechts­krankheiten, Julius Springer, Berlin, Vol. 1, Part 1, p. 434, 1927.

2. BIEDERMANN, W.: Vergleichende Physiologie des Integuments derWirbeltiere, Ergebnisse der BioI. 1: 174, 1926.

3. MIESCHER, G.: Die Chromatophoren in der Haut des Menschen,Arch. f. Dermat. u. Syph. 139: 313,1922.

4. MASSON, P.: Les naevi pigmentaires, tumeurs nerveuses, Ann.d'anat. path. 3: 417,657, 1926.

5. MEIROWSKY, E.: Verstarkung der Pigmentbildung durch oxydiertesAdrenalin (Omega), Arch. f. Dermat. u. Syph. 163: 135, 1931.

77

870 GEORGE MILTON SMITH

6. EWING, J.: The problems of melanoma, Brit. M. J. 2: 852,1930.7. BECKER, S. W.: Cutaneous melanoma: a histologic study especially

directed toward the study of melanoblasts, Arch. Dermat. &Syph. 21:818, 1930.

8. PECK, S. M.: The melanotic pigment in the skin, hair, and eye of thegray rabbit, Arch. Dermat. & Syph. 23: 705, 1931.

9. Foor, N. C.: On the silver impregnation of melanotic tumors, Am. J.Path. 7: 619, 1931.

10. CORDIER, R.: Les pigments melaniques et la melanogenese, Bull.Soc. Roy. d. Sc. med, e nat. de Bruxelles, Nos. 2-7, page 43, 1928.

11. TOLDT, K., JR.: Ueber die flachenhafte Verbreitung der Pigmente inder Haut bei Menschen und Affen, Mitteil. d. anthrop. Gesell.Wien. 51: 160, 1921.

12. !(UKLENSKI, .J.: Ueber das Vorkommen und die Verteilung desPigmentes in den Organen und Geweben bei japanischen Seiden­huhnern, Arch. f. mikr. Anat, 87: 1, 1916.

13. STEINER-WOURLISCH, A.: Das melanotisehe Pigment der Haut bei dcrgrauen Hausrnaus (Mus musculus. L.), Ztschr, f. Zellforsch. undMikroskop. Anat. 2: 453, 192.5.

14. BALLOWITZ, E.: Die Nervenendigungen der Pigmentzellen, cinBeitrag zur Kenntnis des Zusammenhanges der Endveraweigungender Nerven mit dem Protoplasma der Zellen, Ztschr. f. wisscn­schaft. ZooI. 56: 673, 1893.

15. BALLOWITZ, E. : Uber die Pigrnentstromung in den Farbstoffzellcn unddie Kanalchenstruktur des Chromatophoren-Protoplasmas, Arch.f. ges, Physiol, 157: 165, 1914.

16. VON FRISCH, K.: Beitrage zur Physiologie der Pigmentzellen in derFischaut, Arch. f. ges, Physiol. 138: 319, 1911.

17. VON FRISCH, K.: Uber fnrbige Anpassung bei Fischen, ZooI. Juhrb.,Abt. f. allg, Zool. 32: 171, 1912.

18. SOLDAN: Uber die Beziehungen der Pigmentrnaler zur Neurofibroma­tose, Arch. f. klin. Chir. 59: 261, 1899.

In. HEED, H. D., AND GORDON, M.: The morphology of melanotic over­growths in hybrids of Mexican killifishes, Am. J. Cancer 15:1524, 1931.

20. FUKUI, K: On the color pattern produced by various agents in thegoldfish, Folia anat. Jap. 5: 257,1927.

21. SMITH, G. M.: The occurrence of melanophores in certain experi­mental wounds of the goldfish (Carassius auratus), BioI. Bull. 61:73, 1931.

22. MIESCHER, G.: Die Histologie der akuten Rontgendermatitis(Rontgenerythem) , Arch. f. Dermat. u. Syph. 148: 540, 1925.