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THE
EFFECT
EYE
Crll
OF 5-FLUOROURACIL
PIGMENTARY
EPHESTIA
Restwch
ON THE
SYSTEM
IN
K UHNIELLAI
W. MUTH2
Department
of Biology,
University
Received
qf Rochester,
February
Rochester,
N.Y.,
U.S.A.
24, 1964
THE development
Cell Research
37
(30-l)-2902
of the U.S.
Universitgit
Atomic
zu Kiiln.
Energy
Com-
Effect
of 5-fluorouracil
MATERIALS
AND
METHODS
A wild-type
Ephestia
strain, originally
obtained from North Carolina (NCR) and
inbred for 47 and 48 generations
in this laboratory,
was used. The larvae were fed
on yellow cornmeal and raised in rectangular
plastic jars in a constant temperature
room at 2%24C.
In order to obtain pupae of exactly known age, fully grown larvae at the beginning of the prepupal
stage were collected
and transferred
to Petri dishes with a
small amount of cornmeal.
At intervals
of 6 hr these dishes were checked and the
freshly molted pupae removed. They were treated at the following
times:
%nd day: 30-36 and 42-48 hr after
1st duy: 6-12 and 1X-24 hr after pupation;
pupation;
3rd day: 54-60 and 66-72 hr after pupation;
4th day: 78--84 hr after pupation; Sth day: 102-108 hr after pupation;
6th day: 126-132 hr after pupation;
7th day:
150-156 hr after pupation.
5-Fluorouracil
(5-FU) was a gift from Hoffmann-La
Roche, Inc., nutley,
N.J.
Its application
was simple: At the desired age the etherized
pupa was punctured
with sharp watchmaker
forceps between the 5th and 6th abdominal
segment just
above the heart tube, and a small crystal (about $ mm in diameter)
was pushed into
the abdomen. The wound was sealed with hot paraffin. All operations
were done at
10 times magnification
under a stereomicroscope.
Although
the dose of 5-FU administered
in this way was inaccurate,
the experiments yielded precise and completely
reproducible
results without
any variation.
Apparently
the amount
applied constituted
the saturation
dosis with respect to
the phenomena
studied.
In order to estimate the interval from the time of implantation
to the time when
5-FU reaches the sites of action, the following
tests were carried out: crystals were
implanted
into 24-hr-old
pupae and every 15 min thereafter
three animals were
examined.
After 45 min about + to 2 of the crystal was dissolved and after 16 hr
no crystal could be recovered. The distribution
of a dye was used to obtain a rough
estimate of the rate of transport.
Small crystals of fast green, implanted
into 6-12hr-old pupae, led to coloration
of the eyes after 30 min and after 2 hr to coloration
of the wing-lacunae.
In S-day-old pupae, I hr passed until the dye could be demonstrated in the basal parts of the legs (it was not visible in the eyes because of their
dark pigmentation)
and after 4 hr it reached the tips of the wings.
In spite of its obvious disadvantages
this method of application
of the drug was
chosen, because it made possible the application
of 5-FU without
additional
complications
such as osmotic shock, liquid pressure and salt effects, so that the death
rate could be kept down close to zero. After treatment
the pupae remained
in Petri
dishes on filter paper at the same temperature
as before. At the end of the experiment the eyes were drawn, the heads cut off and prepared for paper chromatography.
Five, 10 or 20 heads per group were ground up in 0.2-0.4 ml methanol/HCl
(2 per
cent HCl) and after centrifugation
the extracts were transferred
t.o Whatman
No. 1
filter paper strips and chromatographed
in ascending
direction
with formic acid
(85 per cent)/methanol/HCl
(10 per cent) in ratio 80:15:0.5
[14].
Experimental
Cell Research
37
RESULTS
Cell Research
37
Effect of A-fluorouracil
66-72
78-M
on eye pigmentary
102-
106
system
126 - 132
57
f50-
156
Fig. l.-The
progressive
pigmentation
of the eye. The numbers
on top indicate
the age of the
pupa when 5-FU was implanted.
The upper row of drawings
shows the state of pigmentation
at
the time of treatment;
the lower row shows the state finally
reached.
Cell Research
37
58
11. illufh
Fig. 2.-Development
of normal
(c) 126-132
hr; (d) 174-180
hr.
eye pigment
in the
Ephestia
pupa.
((I) 42-48
hr;
Between
60 and 66 hr the pigmented
area begins to expand beyond this
border and tiny red orange dots appear in the more distal parts. While
these cover more and more of the surface of the eye, the dorso-caudal
part
gradually
turns into a darker red to red-brown
color, a process that subsequently
extends also to the distal area. Between
108 and 126 hr the pigment reaches the anterior edge of the eye. While the most distal part still
darkens,
the eye at 150-156 hr becomes filled all over simultaneously
with
a dark pigment, thus leading to the final homogeneously
black coloration.
On the 8th day these processes which can be observed by surface inspection
Experimental
Cell Research
37
Erect
of 5-fluorourucil
Fig. S.-Result
of implantation
of 10 days. (a) Implantation
hr; (d) implantation
at 54-60
on eye pigmentury
59
system
of 5-F1J crystals
into pupae of known
ages. All observed
at age
at 6-12 hr; (b) implantation
at 18-24 hr; (c) implantation
at 30-36
hr.
are completed.
(For the histological
changes correlated
with the pigmentation, [see 13, 171.)
The effect of 5-FU on the pigmentation
process.-If
applied at early stages,
5-FU blocks the expansion
of the pigmented zone, the level reached depending on the time of implantation
(Fig. 1, lower row and Fig. 3). In general,
the older the animal is at time of implantation,
the farther the pigmentation
does proceed. After implantation
into 6-12-hr-old
pupae the pigment remains
restricted
to the halfmoon-shaped
region in the dorso-caudal
area, but during
Experimental
Cell Research
37
60
W. Muth
Cell Research
37
Effect of 5fluorouracil
on eye pigmentnry
61
system
On chromatograms
of extracts from twenty normal heads component
III
could be observed on the 2nd pupal day, though in very low amounts,
but
during the following days it increased gradually. Component
I was first visible
as a faint red streak on the 4th day, also with subsequent increase in quantity.
The violet pigment 0 did not appear before the 7th day and was present in
Component
TABLE
I.
Normal
day
appearance
of
Formation
by 5-FU
inhibited
before day
2
4
I
0
III
DISCUSSION
From the results presented it appears that 5-FU separates the development
of the eye pigmentary
system into two separate phases. The first phase is
marked
by a spreading process, which enables the cells taken over by it to
Experimental
Cell Research
37
Cell Research
37
Effect of 5fluorourncil
on eye pigmentnry
system
63
SUMMARY
Cell Research
37
hr after pupation
blocks
a spreading
process
which
is necessary
for the
synthesis
and deposition
of the two pigment
components
which
appear
first
in development.
Treatment
with 5-W up to the fourth
tlay after pupation
suppresses
completely
the formation
of a third
component
of the pigment,
while
after this time S-FU has no effect on the formation
of the eye pigments,
even though
the third pigment
component
appears
only at the 7th clay after
pupation.
The results
are discussed
with respect
to possible
mechanisms
untlerlping
the effects of 5-FU on the processes involved
in pigment
formation.
I wish to express my gratitude to Dr Ernst W. Caspari for the hospitality
I received
in his laboratory
and for his continued
encouragement
during the course of these
investigations,
and to Mr Nicholas
Cohen for his help in the preparation
of the
photographs.
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Experimental
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