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The use of ultraviolet light to induce melanogenesis in the epidermis of the rhesus monkeyAn ultrastructural and biochemical study.

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The Use of Ultraviolet Light to Induce Melanogenesis
in the Epidermis of the Rhesus Monkey: An
Ultrastructural and Biochemical Study ' f 2
Department of Cutaneous Biology, Oregon Regional Primate Research
Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005
The general body epidermis of the rhesus monkey (Macaca
mulatta) contains no discernible melanocytes, but after repeated ultraviolet
irradiation DOPA-positive melanocytes appear and increase numerically up to
30 exposures. With continued irradiation, however, the number again declines.
Experiments to determine how melanogenic activity, assayed by the incorporation of labeled DOPA or tyrosine, is related to DOPA positivity indicated that
biochemical activity corresponded to the histochemical pattern. Ultrastructural
studies demonstrated that after the exposure to ultraviolet light, a pool of indeterminate cells in the skin of rhesus monkeys developed into melanocytes. The
melanosomes formed by these cells, however, differed from the eumelanin melanosomes described in other species; they had no internal filamentous matrix
with periodicity but appeared similar to phaeomelanin melanosomes. Long term
ultraviolet light irradiation may damage keratinocytes and render them incapable of phagocytizing melanosomes.
The pigmentary reaction to chronic
ultraviolet exposure has already been described in the rhesus monkey (Macaca
mulatta) by Erickson and Montagna ('75).
They have shown that although the general body epidermis of the rhesus monkey
has no discernible melanocytes, histochemically demonstrable DOPA-positive melanocytes appeared after sequential ultraviolet
(UV) irradiation, increased to peak numbers after 30 exposures, then steadily declined to basal level.
The experiments reported here were designed to determine how ultrastructural
features and melanogenic activity in skin
sequentially irradiated with UV light are
related to DOPA positivity.
mals received no irradiation. The spectral
region of the lamp was 185 nm to 313 nm.
Before irradiation, biopsies were removed
from all animals, and 14 times during the
exposure period nine biopsies were removed from three randomly selected animals (Erickson and Montagna, '75).
Electron microscopy
Blocks of tissue, 1 to 2 mm2, were fixed
directly at 4°C in buffered 1% OsOc (pH
7.2) (Palade, '52) or 2.0% paraformaldehyde plus 2.5% glutaraldehyde solution in
0.03 M Millonig's phosphate buffer (pH
7.2) (Russell, '72), washed in 0.16 M
phosphate buffer then postfixed in 1%
Os04 in 0.08 M phosphate buffer. Tissues
were dehydrated through graded ethanol
to propylene oxide and embedded (Spun,
Irradiation methods
These studies were conducted on eight
adult male rhesus monkeys (Macaca mulatta). After the hair had been removed,
the thorax and abdomen of six animals
were exposed five times per week to 7.0 X
lo5 pw/cm2 of UV light (Hanovia Aero
kromayer UV light) for 83 doses; two ani-
Received July 7, '75. Accepted Oct. 7, '75.
1 Publication No. 818 of the Oregon Regional Primate Research Center supported in part by Public
Health Service, National Institutes of Health Grants
RR00163 of the Animal Resources Branch, and AM
08445 of the National Institute of Arthritis and Metabolic Diseases.
ZPresented. in uart. at the 87th Annual Session of
the American Association of Anatomists, Cleveland,
Ohio, 1974. (Anat. Rec., 178: 351, 1974).
3 Present address: Department of Human Anatomy,
University of California. School of Medicine, Davis,
California 95616.
ANAT. REC., 184: 637-646.
'69). One-micron sections were stained
with toluidine blue. Sections for electron
microscopy were stained with lead citrate
and uranyl acetate and viewed with a
Philips 200 electron microscope operating
at 60 KV.
Biochemical studies
After 10, 13, 18, 23, 28, 32, 38, 43 53,
and 68 exposures, four 1 x 3 cm elliptical
biopsies were taken from two randomly
selected animals, one of the two nonirradiated controls and one of the six irradiated animals,
Tyrosinase activity was measured with a
modified technique described by Kitano
and Hu ('71) for determining melanin
synthesis. Epidermal sheets were prepared
by incubating full-thickness biopsies in 2 N
NaBr for one and one-half hours at 37°C
and separating the epidermis from the
dermis. The sheets were blotted dry,
weighed, and incubated in TC Hanks solution (Difco Labs) for 30 minutes at 37°C.
For a control,
M 1-phenyl-2-thiourea
was used. This compound inhibits the deposition of melanin on the internal matrix
of the melanosome by complexing with the
copper present in tyrosinase. To the solution, 0.5 &i/ml, DL-3, 4-dihydroxyphenylalanine-2-C" (DOPA) (New England Nuclear, spec act 9.31 mCi/mM) was added
for two hours at 37"C, after which the
samples were incubated in a 0.1% DOPA
solution for 15 minutes. The samples labeled with c'"tyrosine were incubated with
0.5 pCi/ml L-tyrosine-l-Ci4 (New England
Nuclear, spec act 53.5 mCi/mM) for two
hours at 37"C, then postincubated in 0.1%
aqueous solution of tyrosine. For a control,
1-phenyl-2-thiourea and/or 100 nig/ml
puromycin were added. (The latter was
used to inhibit gener a1 protein synthesis. )
The epidermal sheets were homogenized
in a blender and trichloracetic acid (TCA)
was added to the homogenate for a final
TCA concentration of 5% ; samples were
placed in an ice bath overnight. The resulting precipitates were collected on millipore
filters (HAWP25) and washed first with
cold 5% TCA and then absolute isopropanol. Dry filters were placed in glass
scintillation vials containing toluene and
omnifluor (New England Nuclear) and
counted in a liquid scintillation spectrom-
eter. The dermis was processed exactly
like the epidermis except that before
homogenation it was frozen in liquid nitrogen and pulverized in a cold stainless-steel
The rate of incorporation of C14-DOPA
or Ci4-tyrosine puromycin into ultraviolet-stimulated epidermis was compared
with that of controls (the above labeled
compounds plus phenylthiourea) . The difference is reported as the rate of melanin
Ultrastructural study
Ultrastructural investigations confirmed
the earlier histological observations (Erickson and Montagna, '75) that no melanocytes were present in the general body epidermis of the nonirradiated adult rhesus.
Most epidermal cells were ker atinocytes ,
but two types of dendritic cells were also
present, Langerhans cells and indeterminate cells. The former were found in all
strata of the Malpighian layer but most frequently in the spinous layer. These cells
contained microfilaments and had irregular, indented nuclei and characteristic
Langerhans granules which, like the electron-opaque bodies, were randomly dispersed (fig. 1). Since acid phosphatase activity has been demonstrated in these
latter structures (Rowden, '67; Wolff, '67),
they may be lysosomes. Like other dendritic cells, the indeterminate cells possessed similar features, e.g., microfilaments which do not aggregate into bundles
but lack the defining cytoplasmic organelles, Langerhans granules or melanosomes, and desmosomes. These cells usually had indented nuclei but lacked the
typical electron-opaque bodies seen in the
Langerhans cells. Vesicles were sometimes
Fig. 1 A Langerhans cell in the epidermis of
nonirradiated skin. The surrounding keratinocytes contain tonofibrils whereas the Langerhans
cell has distinct microfilaments (MF). Characteristic of this cell is the Langerhans granules (LG).
Numerous lysosomes ( L ) or electron opaque
bodies are present. x 30,000.
Fig. 2 The indeterminate cell in the epidermis of nonirradiated skin. Neither Langerhans
granules nor melanosomes are present. This cell
lacks the typical electron opaque bodies shown
in the previous figure but contains well-defined
microfilaments (arrows 1. x 19,000.
observed in the cytoplasm, more often in
the basal layer, only occasionally in the
spinous layer. Though less common than
Langerhans cells, these cells constituted a
sizable component of the normal epidermis in the rhesus monkey (fig. 2).
After four irradiations, a few active
melanocytes with all stages of melanosome
formation were visualized. Most developing
melanosomes (stages I and 11) lacked a n
organized internal matrix, but a few had a
filamentous network (fig. 3). Melanization
seemed to derive from the deposition of a
central core of flocculent material which
gradually filled the membrane-limited vesicle until it became uniformly electron
opaque (fig. 3 ) . After four irradiations, a
few fully developed melanosomes (stage
IV) were seen. During this period, very
few single melanosomes and no membrane-bounded complexes were transferred
to the surrounding keratinocytes. Stimulated melanocytes had an active Golgi apparatus and an extensive rough endoplasmic reticulum, Numerous small vesicles
(50-80 n m ) were also dispersed throughout the cytoplasm. Mitotic figures were observed in the keratinocytes of the basal
layer but not in the developing melanocytes or indeterminate cells.
After 15 exposures, the increased population of melanocytes contained all stages
of melanosome formation, but at this stage
developing melanosomes were less numerous than after four exposures. A small
number of indeterminate cells had active
Golgi zones and many small vesicles (40150 nm). The stimulated melanocytes
occasionally contained partially melanized
melanosomes or clumps of smaller particles which resembled melanosomes and
were similar to phaeomelanin (fig. 4).
After 25 exposures, a few melanosomes
were transferred to the surrounding keratinocytes, the rest remaining near the
periphery of the melanocytes. At peak activity (30 exposures), the melanocytes contained fully developed melanosomes (stage
I V ) but no clearly discernible early-stage
melanosomes (stages I and 11). The rough
endoplasmic reticulum was not as active
as before; often very few or no Golgiassociated vesicles could be seen (fig. 5).
After 37 exposures, the melanocytes contained only a few melanosomes (fig. 6)
and showed no signs of extensive activity;
however, no signs of damage were apparent, In specimens which had received additional irradiation, the population of indeterminate cells seemed to be increasing.
These cells were quite similar to those in
untreated epidermis except that they had
more microfilamen t s.
Biochemical study
In general, the uptake of C'*-DOPA or
C'*-tyrosine plus puromycin by melanosomes was similar (fig. 7): with sequential irradiation, the incorporation of melanin precursor rose to a peak; with
continued irradiation, it decreased to a
basal level, The maximum uptake and the
length of time it took to incorporate them
differed for both compounds, but both had
a single peak. In addition, phenylthiourea
did not completely inhibit melanin synthesis. In figure 8, only the rate of melanin
synthesis (i.e., the rate of incorporation of
C"-DOPA, minus the rate of CI4-DOPAplus
phenylthiourea) is given. It indicates a
pattern similar to that shown by the histochemical data, i.e., an increase to Deak
activity followed by an equally rapid decrease.
The technique used for determining the
rate of melanin synthesis is probably sensitive enough to demonstrate melanogenic
activity when small numbers of melanocytes are present. But one problem with
this technique is that phenylthiourea does
not completely inhibit tyrosinase activity
even though Lerner and his co-workers
('50) reported it to be the most effective
inhibitor available. Kitano and Hu ('71)
also reported that it is virtually impossible
to abolish tyrosinase activity with inhibitors without simultaneously imparing protein synthesis. Protein synthesis, but apparently not melanoprotein synthesis, is
inhibited by puromycin. Moreover, the cessation of protein synthesis does not affect
Fig. 3 A dendrite of a melanocyte. Several
stages of melanosomes (11-IV) are present. The
early stage melanosomes generally lack an organized internal matrix although occasional filamentous networks are seen (single arrow). Melanization appears to be a deposition of a flocculent material (double arrows). A few completely
developed melanosomes ( I V ) are found within
the melanocyte. x 35,000.
Figure 3
64 1
Fig. 4 A n epidermal melanocyte after 21 exposures. Some melanosomes (arrow), which appear
morphologically similar to phaemelanin, exhibit incomplete melanization. x 14,000. Insert x 36,000.
tyrosinase activity (Kitano and Hu, '71).
In my experiments, the level of inhibition
by phenylthiourea, which was of the same
magnitude as that reported by Kitano and
Hu ('71), varied according to the number
of ultraviolet exposures, but the activity
was never completely inhibited.
The pattern of biochemical activity parallels that of histochemical data. Why the
peak incorporation of C14-DOPA or of
C"-DOPA plus inhibitors occurs at 33 days
instead of at 28 days when the difference
between the two is maximal is not known.
This part of the study demonstrates that
there is no compensation on the part of
enzyme activity, i.e., when the number of
morphological units decreases, so does the
total amount of enzyme activity.
There is no consensus about the origin
of DOPA-positive cells after UV irradiation. After irradiating several species of
vertebrates, a number of investigators
(Sato and Kawanda, '72a, '72b; Snell, '63;
Wolff and Winkelmann, '67) concluded
that the DOPA-negative dendritic cells at
the dermo-epidermal junction are amelanotic melanocytes and that the increase
in DOPA-positivity is partly due to the activation of these normally inactive cells.
Sat0 and Kawanda ('72a) based their conclusion on an experiment in which 1.1%
of the DOPA-positive cells were labeled
with H3-thymidineduring ultraviolet irradiation. On the basis of mitotic figures and
incorporation of H3-thymidine within the
melanocytes, Quevedo et al. ('63), however, concluded that division accounts for
at least part of the population increase in
the melanocytes of irradiated pedal skin.
Mishima and Widlan ('67) substantiated
the latter hypothesis.
In my experiments, exposure to ultraviolet light stimulated a pool of indeterminate cells in the epidermis of the hairy
skin to form melanocytes. This conclusion
is based on the observation that significant
numbers of indeterminate cells are found
in nonirradiated skin and in skin biopsied
Fig. 5 A melanocyte with the surrounding keratinocytes after 30 exposures. The melanosomes
are fully developed. Although two stage I11 melanosomes are present (arrows), no earlier forms are
evident. x 24,000.
Fig. 6 A melanocyte in the basal layer of the epidermis after 37 exposures. Only a few melanosomes are present (arrow). x 19,000.
after peak melanogenic activity, but virtually none at the peak of melanogenic
Other authors have arrived at similar
conclusions. Zelickson and Mottaz ('68)
suggested that indeterminate cells represent either a form of premelanocyte in
which melanin synthesis can be induced
or an effete melanocyte which has ceased
to function. If the latter, then indeterminate cell should also be found in the upper
layers of the epidermis; but as I have
shown in these studies, they axe predominantly in the lower layers. Zelickson and
Mottaz ('68) also thought that indeterminate cells are undifferentiated cells that
give rise to Langerhans cells, melanocytes,
or completely unrelated cells. Later they
reported that a decrease in indeterminate
cells as well as an increase in the mitotic
activity of existing melanocytes accounted
in their study for the increase in melanocytes (Zelickson and Mottaz, '70). After
two weeks of daily ultraviolet irradiation,
no Langerhans or indeterminate cells were
observed. Another group of investigators
(Tsuji et al., '69) hypothesized that indeterminate cells are undifferentiated cells
which may give rise to another epidermal
dendritic type cell or that melanocytes and
Langerhans cells can transform into each
other through indeterminate cells. The indeterminate cell may also represent a form
of premelanocyte in which melanin synthesis can be induced, but whether any
Langerhans cell-melanocyte transformation can occur is doubtful. On the basis of
morphological and experimental data, the
latter two are probably separate cell populations in the epidermis (Breathnach et
al., '68); therefore, the transformation
hypothesis is not cogent.
Although these experiments do not explain what happens to the formed melanosomes and why the melanocytes suddenly
stop forming them, some conclusions can
be drawn. Melanocytes, melanosomes, and
keratinocytes are closely related (Fitzpatrick and Breathnach, '63; Hadley and
Quevedo, '66, ' 6 7 ) , and the latter probably
Melanin Synthesis in Melanocytes of
Irradiated Skin
-___-_C'*- DOPA + Ph.
- C''-Tyrosine+Pu.
700 -
300 200
Number of Ultraviolet Light Irradiations
Fig. 7 Uptake of labeled melanin precursors in the epidermis after exposure to 7.0 x 105
pw/cm2 uv light. Bmchets indicate standard error of the mean. Ph., phenylthiourea, h,
Melanin Synthesis in Melanocytes of Irradiated
Skin (C'=DOPA)-(C"-DOPA + Ph.)
Number of
Ultraviolet Light Irradiations
Fig. 8 Rate of melanin synthesis after sequential daily irradiation. Data are extrapolated from
figure 7.
phagocytize portions of the melanin-laden
dendrites of melanocytes (Fitzpatrick and
Breathnach, '63; Cruickshank and Harcourt, '64). Thus, the rate of melanin
synthesis within melanocytes may be regu-
lated by a feedback mechanism which depends upon the rate at which melanosomes
are removed by keratinocytes. This mechanism may function in man, but probably
not in the rhesus monkey in which melanosomes are not rapidly transferred to
surrounding keratinocytes. In irradiated
rhesus skin, no melanosomes or melanocytes pass into the dermis and few or none
remain in the epidermal melanocytes. Actually, melanosomes are transferred to
keratinocytes but probably at such a slow
rate that any new melanosome synthesis
is inhibited. This may be a defect in the
ability of the keratinocytes to take up the
Several other investigators have reported
the inability of keratinocytes to take up
melanosomes under various pathological
conditions. Mitchell ('63) reported that
after prolonged solar irradiation of Caucasian skin, melanocytes seemed unable to
transmit pigment to the keratinocytes, but
very dark skin, like that of aborigines, did
not appear to be damaged even by years
of exposure to the sun (Mitchell, '68).
Solar degeneration in xeroderma pigmentosum also prevents pigment transfer from
occurring normally (Olson et al., '70). In
this condition, some keratinocytes contained little melanin but others showed
higher than normal amounts. Likewise,
the passage of melanosomes to keratinocytes was inhibited after minor trauma,
which had caused intercellular edema.
Mottaz et al. ('71) thought that this condition resembles atopic dermatitis or that
it is another example of the inability of
melanosomes to be transferred to keratinocytes.
As I have reported earlier (Erickson
and Montagna, ' 7 5 ) , UV light may cause
some physical-chemical changes in the
epidermis such as cell injury since after
successive periods of irradiation and nonirradiation, reirradiation causes active melanocytes to reappear. The exact nature of
this injury is difficult to determine until
better techniques have been developed.
I would like to express my gratitude to
Drs. William Montagna and Mary Bell for
their generous assistance and advice, and
to Drs. Funan Hu and W. H. Fahrenbach
for their helpful suggestions.
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