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The radiomimetic effect of neotetrazolium chloride on the skin of mice.

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T H E RADIOMIMETIC EFFECT OF NEOTETRAZOLIUM CHLORIDE ON T H E SKIN
O F MICE
THOMAS S. ARGYRIS'
Department of Zoology, Syracuse Unizhersity, Syracuse 10, N.P.
and Department of Anatomy, Harvard Medical School,
Boston, Mass.
TWELVE FIGURES
INTRODUCTION
At the injection site of neotetrazolium chloride (N.T.) in
colored mice, Antopol, et al. ('50) observed greying of hair.
They suggest that this greying might indicate that neotetrazolium is a radiomimetic substance, since x-rays are known
to produce greying (Chase, '49). A further investigation
of the effects of N.T. on the skin of mice can perhaps answer
some questions of radiomimicry in general. Such a study is
pertinent because most of the investigations on the biological
effects of radiomimetic agents are limited to the analysis
of one o r two parameters; in the cell, tissue, or organism under
investigation (Bacq, '55; Boyland and Sargent, '51). The
skin has a variety of integrated cell populations forming
sub-organ units, e,g., hair follicles, epidermis, sebaceous
glands, dermis, subcutaneous tissue, the composition and
function of which vary at different stages of the hair growth
cycle (Chase et al., '53). Many of the sequential adaptive
responses of these integrated skin sub-units are known for
various doses of x-irradiation and f o r different stages of
the hair growth cycle (Chase, '49; Chase and Rauch, '50;
The experimental portion of this work was done while a U.S.P.H.S. Postdoctoral
fellow a t Harvard Medical School.
697
698
THOMAS S. ARGYRIS
Argyris, '54). Therefore, an accurate comparison can be
made between the biological effects of N.T. and x-irradiation,
on a number of levels of organization and in a definite timespace relationship.
MATERIALS AND METHODS
Approximately 150 C,, Black mice of both sexes ranging
between three and 6 months of age were used. All animals
were kept in plastic cages and offered Purina laboratory chow
and water ad libiturn.
In order to be certain of the stage of hair growth, the
entire dorsum of each mouse was plucked when the skin was
in the resting phase of the hair growth cycle. Plucking initiated hair growth in the plucked area only (Chase, et al.,
'51). Mice were injected either at 9 days after plucking, or
at 21-25 days after plucking. The former group represented
mice with skin in the growing phase of the hair cycle and
the latter, mice with skin in the resting phase of the hair cycle.
Group I
Group
Group
Group
Group
Animals received 0.5 cc of a 0.1% solution of N.T.2 in saline.
~ a 0.1% N.T.
I1 Animals received 0 . 5 of
in saline and the resulting bleb of fluid
was massaged into the skin.
111 Crystals of N.T. were introduced subcutaneously by means of a trocar.
IV Animals received 0.5 cc distilled water
subcutaneously.
Animals received 0.5 cc distilled water
V
and the bleb of fluid massaged into the
skin.
For Groups I and I1 appropriate saline controls were
devised. For Group I1 a trocar without N.T. was introduced
subcutaneously and then removed.
a Purcliased from General Biochemicals, Inc., Laboratory Park, Chagrin Falls,
Ohio.
RADIOMIMICRY A N D MOUSE SKIN
699
All injections were done under ether anesthesia. Animals
were checked daily. A record was kept of their general
condition, and the condition of the skin, including the stage of
the hair cycle.
RESULTS
I. S y s t e m i c effects
Mice injected with 0.5cc of 0.1% N.T. o r with crystals of
N.T., (Group I, 11, and 111) become very quiet within 15
minutes. They remain quiet f o r approximately 15 hours.
During this period, breathing is deep and occasionally arhythmic. By 24 hours the mice recover. No systemic effects are
noticed after the injection of distilled water or saline (Groups
IV, V, and Controls).
I I . Skin effects
Gradual hair loss occurs within 6-14 days in skin with
resting follicles injected with 0.5 cc of N.T. (Group I). An
area of skin of approximately 30mm2 is epilated, but there
is some variation (fig. 1). Within the next few days the
epilated area becomes rough and crusty. Occasionally a scab
is seen in the center of the denuded area indicating further
damage. Following this, the surface of the skin again becomes
smooth. Within a few days a rim of white hairs emerges
from the periphery of the wound giving a greying appearance
(fig. 2). I n addition, the surrounding normal resting hairs
are induced to grow, indicated by a thin circular line of black
growing hair follicles circumscribing the regenerating wound.
Greying is fully evident within 21-24 days after injection of
the N.T. The area of greying is about the same size as the
original area of hair loss although there is less hair than in
the normal control area.
Skin with growing follicles injected with 0.5 cc of 0.1%
N.T. (Group I), exhibits an abrupt hair loss within one t o
two days after injection (fig. 3). Moreover, the area epilated
is always at least two to 4 times that seen in treated skin with
700
THOMAS S. ARGYRIS
resting hair follicles, using the same dosage of N.T. During
the next few days the center of the wound acquires a lead
colored appearance which occasionally is almost black, and
may show a crust or scab. Following this the wound becomes
progressively smoother and by 17 days after N.T. injection
the entire epilated surface is smooth. By 23 days after injection, regeneration of white hairs is seen around the periphery of the wound surrounded by a thin line of normal
pigmented growing follicles. However, the area of greying
is much smaller than the original area of hair loss. I n some
cases, greying is almost unnoticeable (fig. 4).
Since it may be argued that part of the damage to the skin
of the mice is due to the bleb produced by the subcutaneous
injection of the N.T., animals were injected with 0.5cc of
a 0.1% N.T. solution and the fluid massaged into the skin
(Group 11).
Skin damage and repair is the same as that seen in animals
in which the injected fluid is not massaged into the skin (Group
I). Resting hair follicles lose their hairs gradually within
7-8 days, and greying is apparent by the 20 to 23rd day
after treatment. Growing follicles lose their hairs abruptly
within two days after treatment, and regeneration of new
hairs is apparent by 20-27 days. As before, in mice with resting hair follicles, the area of greying is about the same as
the area of hair loss (fig. 5), and in skin with growing hair
follicles the amount of greying is much less than the amount
of hair loss (fig. 6).
Saline controls in either Group I or 11, show no damage
irrespective of whether the skin is in the growing or resting
phase of the hair cycle at the time of injection.
To be certain that the damage is due to the crystals of
neotetrazolium only, a series of animals were injected subcutaneously with a small amount of N.T. crystals (Group 111).
Again the results are the same. Hair loss is gradual in
skin with resting follicles, beginning usually at 7-8 days after
treatment, and the pattern of damage produced is similar
to that above (fig. 7). Regeneration of white hairs is evident
RADIOMIMICRY AND MOUSE SKIN
701
by 24 days (fig. 8). The area of greying is about the same
as the area of initial hair loss. Abrupt hair loss is evident
within one to two days in skin with growing follicles (fig. 9).
Damage and hyperpigmentation are present as in Group I.
Greying occurs by the 16th to 23rd day and is much less than
the area of initial hair loss (fig. 10). The sham insertion of
a needle subcutaneously does not result in any hair loss or
greying.
Because it has been reported (Boyland and Sargent, '50)
that the intradermal injection of distilled water also produces
greying, a series of mice with resting or growing hair follicles
were subcutaneously injected with 0.5 cc of distilled water.
I n some cases the bleb formed was left alone (Group IV) and
in others the bleb was reduced by massaging with the fingers
(Group V) . With or without the formation of a bleb, the subcutaneous injection of 0.5 cc distilled water does not produce
hair loss or greying in skin with either growing (fig. 11) or
resting follicles (fig. 12).
DISCUSSION
Neotetrazolium chloride injected subcutaneously causes hair
loss and subsequent greying in skin with resting or growing
follicles. Hair loss occurs later in skin with resting follicles
than with growing follicles. Moreover, the amount of hair loss
is much less in skin with resting follicles than in skin with
growing follicles. Greying, however, is greater in skin with
resting follicles as compared to skin possessing follicles even
though the skin in the growth phase loses many more hairs
than the skin in the resting phase. Thus, there is an inverse
correlation between the amount of hair loss and the degree
of eventual greying.
The character of the wound is the same irrespective of
the condition of the skin at the time of treatment. Usually
wounds show a crust or scab, I n addition, wounds in skin with
growing follicles are pigmented.
These results are similar to those produced by an x-irradiation of approximately 1500r. Chase ('49) and Chase and
702
THOMAS S. ARGYRIS
Rauch ('50) have shown that with doses approaching 1500r
skin with either growing or resting follicles is epilated. Not
only is hair loss abrupt and more extensive in skin with
growing follicles but it also occurs earlier. In contrast, the
hair loss which occurs in resting skin begins at approximately
14-21 days and is gradual. A crust is exhibited by all wounds
and skin with growing follicles is hyperpigmented. Hair
regeneration occurs at approximately 30 days after irradiation, irrespective of the stage of the hair cycle of the skin at
the time of irradiation. As after treatment with N.T., there
is more subsequent greying of hairs from skin treated in the
resting phase than from skin treated in the growth phase,
A comparison of the effects of N.T. and x-irradiation might
leave the impression that the timing of some of the principal
events is not similar. With N.T. treatment growing follicles
lose their hairs within 2 days and resting follicles within 7-8
days. After irradiation, however, skin in the growth phase
does not shed its hairs until 4-6 days, and resting skin is
often not fully epilated until 21 days. However, the ratio
obtained by dividing the time of hair loss in growing skin
by that in resting skin, either after x-irradiation or N.T.
treatment, is approximately $. Also, corresponding to the
earlier hair loss after N.T. administration, hair regeneration
occurs earlier, being evident after 23 days as compared to
approximately 30 days for x-irradiation. Therefore, the time
between hair loss and greying is still approximately the same.
It may be argued that although N.T. is radiomimetic on a
gross level, a study of the microscopic changes might reveal
that they are different from x-irradiation. Preliminary evidence (Argyris, '56) however, suggests that the principal
sequential microscopic events are the same.
This report is not the first to note greying after t,reatment
of mouse skin with chemicals. Boyland and Sargent ('51)
have investigated several chemical substances as to their
abilities to produce greying. They suggest that a solution of
5 X 1 0 P M of nitrogen mustard produces the same amount
RADIOMIMICRY AND MOUSE SKIN
703
of greying as a mean dose of 800r. Because of the lack of
attention to hair cycles it is difficult to evaluate the data,
since skin with growing follicles show epilation and little subsequent greying and skin resting follicles will show less
epilation and more greying.
Greying, hair loss, or hyperpigmentation taken singly are
not sufficient criteria for radiomimicry. There are many substances which produce greying, even permanently, which are
not radiomimetic when viewed from the point of view of the
pattern of damage they produce. For example, transplantation (Butcher, '45 ; Reed and Henderson, '40), freezing
(Taylor, '49), and mechanical injury from wounds (Chase,
'56) can produce greying. Even distilled water is claimed
to produce greying in C,, mice (Boyland and Sargent, '51),
although in our hands distilled water, even in larger doses,
does not result in greying. Hair loss cannot, by itself, be
used as a criterion for radiomimicry since it may result from
vitamin or metal deficiencies (Lorincz, '54) or after treatment
by a variety of chemical substances (Chase and Montagna,
'51 ; Rauch, '52). Finally hyperpigmentation, as seen grossly,
cannot be considered radiomimetic. Quevedo ( '56) has shown
that hyperpigmentation may result from methylcholanthrene
application, a substance the effects of which on skin are opposite to those of x-irradiation (Argyris, '52; Chase and Montagna, '51). Permanent greying preceded by hair loss also
is not a sufficient criterion for radiomimicry since transplantation (Butcher, '45 ; Reed and Henderson, '40), and
freezing (Taylor, '49) can result in hair loss followed by
greying.
Hair loss, hyperpigmentation (in skin with growing follicles), and greying when taken together, are quite specific
for radiation and radiomimetic substances as N.T. This is
especially true if not only the three parameters are considered,
but in addition, the timing and integrated response pattern
of these parameters is assayed.
704
THOMAS S. ARGYRIS
SUMMARY
The effects produced by the subcutaneous injection of neotetrazolium chloride (N.T.) into the dorsum of C,, Black
mice, with hair either in the growing or resting phase of the
hair growth cycle, have been investigated.
Skin with growing hair follicles loses its hair within two
days whereas skin with resting follicles is not epilated until
approximately 7-8 days after treatment. The area of hair
loss in skin with growing hair follicles is much larger than
in skin with resting follicles.
Epilation is followed by the appearance of a crust or scab
on the denuded area, irrespective of the stage of the hair
cycle. Skin with growing follicles in addition exhibits a lead
colored appearance due to hyperpigmentation of the skin.
Hair regeneration is evident after approximately 23 days.
The regenerated hairs are either black or white. The number
of regenerated white hairs is much smaller in skin with growing hair follicles than in skin with resting follicles, even
though the area of hair loss is much larger.
This pattern of damage characterized by an inverse correlation between the amount of hair loss and subsequent greying, and hyperpigmentation in skin with growing follicles, is
similar to damage produced by x-irradiation of approximately
1000-1500 r.
Although there are a number of experimental situations
which may result in greying, hair loss, or hyperpigmentation,
or hair loss accompanied by greying, no treatment other than
x-irradiation or N.T. administration results in all three, i.e.
hair loss, hyperpigmentation, and greying, and in the pattern
observed.
It is suggested that the evaluation of whether or not a
substance is radiomimetic should not be based on its ability
to mimic single biological effects produced by x-irradiation,
but should produce the same pattern of damage, involving
a number of parameters.
RADIOMIMICRY A N D MOUSE SKIN
705
LITERATURE CITED
ANTOPOL,W., S. GLAURdCH AND L. GOLDMAN1950 The use of neotetrazolium
as a tool in the study of active cell processes. Trans. N. Y. Acad. Sci.,
1 2 : 156-160.
ARGYRIS,1’. S. 1952 Glycogen in the epidermis of mice painted with methylcholanthreiie. J. h’at. Cancer Inst., 12: 1159-1166.
1954 The relationship between the hair growth cycle and the
response of mouse skin to x-irradiation. Am. J. Anat., 94: 439-472.
1956 To what extent must a chemical substance niirriic radiation
to be considered radiomimetic. Aiiat. Rec., 164 : 253-254.
BACQ,Z. M., AND P. ALEXANDER1955 E’uIidamentals of Radiobiology. Butterworth’s Scientific Publications. London. Chap. V I I , 190-208.
BOYLAND,
E., AND S. SARGENT
1951 The local greying of hair in mice treated
with x-rays and radiomimetic drugs. Brit. J. Cancer, 5 : 433-440.
BUTCHER,
E. 0. 1945 Pigmentation of hair on transplanted skin i n hooded rats.
Arch. Derm. and Syph., 56: 347-350.
CHASE, H. B. 1949 Greyiiig of hair. I. Effects produced by single doses of
x-rays 011 mice. J. Morph., 84: 57-80.
1956 Personal communication.
CHASE,H. B., AND H. RAUCH 1950 Greyiiig of hair. 11. Respoiise of individual
hairs i n mice to variations in x-irradiation. J. Morph., 67: 381-392.
CHASE,H. B., AND W. MONTAGNA1951 Relation of hair proliferation t o damage
induced in the mouse skin. Proc. Soc. Exp. Biol. and Med., 7 6 : 35-37.
CHASE, H. B., H. RAUCHAND v. w. SMITH 1951 Critical stages of hair development aiid pigmentation in the mouse. Physiol. Zool., 2 4 : 1-8.
A N D J. I). MALONE 1953 Changes in tho skiii iii
CHASE, H. B., W. MONTAGNA
relation to the hair growth cycle. Aiiat. Rec., 116: 75-82.
GEARY,J. R. 1952 Effect of roentgen rays during various phases of the hair
cycle of the albino rats. Am. J. Anat., 9 1 : 51-106.
LORINCZ,
A. L. 1954 Physiology and Biochemistry of the Skin. Ed. S. Rothman,
University of Chicago Press, Chicago. Chap. X X V I I , 662-698.
QUEVEDO,
W. C. 1956 Piginent cell behaviour and hair growth cycles: a s influenced by gene substitutions and experimentally induced changes i n the
skin of mice and rabbits. Thesis - Brown University, Ph.D. June.
RAUCH,H. 1952 The effect of topical application of chemical agents on hair
development. Physiol. Zool., $5 : 268-272.
REED, 8. C., AND J. M. HENDERSON
1940 Pigment cell migration i n mouse
epidermis. J. Exp. Zool., 85: 409-418.
TAYLOR,A. C. 1949 Survival of r a t skin and changes i n hair pigmentation
following freezing. J. Exp. Zool., 110: 77-112.
PLATE 1
EXPLANATION OF BTGURES
All mice are one-lialf norinal size
Skin with restiiig follicles treated with 0.5 em3 of neo trazoliuni chloritlcl
(N.T.) subcutaneously. Fourteen days a f t e r treatment, note area of hair loss.
Greying ill skin with resting follicles 28 days a f t e r treatnieiit with N.T.
Animals iiitlicate the lower mtl upprr limits of greying produced wit.h 0.5 cir?
of N.T.
Hair loss in mouse wit11 growing hair follicles, 6 days aft,cr i n j
0.5 0111~ of N.T.
Greying, 41 days a f t e r ailmiiiistratiori of 0.5 em3 of N.T. t o mice with gro\ring
hair follicles. Note almost complete absence of greying in two of the niioe.
Compare with figure 2.
Greying in skin with resting follicles. The injected N.T. has been tilassaged
into the skim of the two animals in the right haiid group. The single animal
on the left is one in which the N.T. has n o t been mass:iged into the skin.
Greying in mice with growing follicles 40 days after N.T. treatment. Tlic
N.T. has beoil massaged into the skin.
706
RADIOMIMICRY AND MOUSE SKIN
PLATE 1
THOMAS S. ARGYRIS
707
PLATE 2
EXPLANATION OF FIGURES
All mice are one-half normal size
7
Arrow indicates the area of hair loss and wound 10 days after the injection
of N.T. crystals in skin with resting follicles. Lower portion is clipped to
check the hair cycle.
8
Greying in mice with resting follicles treated with N.T. crystals. Right mouse
is control. Control area is clipped to show absence of damage.
9
Hair loss in skin with growing follicles three days a f t e r treatment with
crystals of N.T.
10 Regeneration of hair a f t e r treatment with crystals of N.T. in skin with
growing follicles. Greying is barely perceptible.
11 Absence of greying in skin with growing follicles treated with distilled water.
Right animal, distilled water was massaged into the skin.
12
Absence of greying in mice with resting hair follicles treated with distilled
water. Eight animal. distilled watcr was massaged into the skin.
708
RADIOMIMICRY A N D M O U S E SKIN
THOMAS S. ARGYRIS
PLATE 2
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