the effect of 5-fluorouracil on the eye pigmentary system in ephestia kuhniealla

Experimenid Crll Restwch 37, S&6 I (1965)

THE EFFECT OF 5-FLUOROURACIL ON THE

EYE PIGMENTARY SYSTEM IN

EPHESTIA K UHNIELLAI

W. MUTH2

Department of Biology, University qf Rochester, Rochester, N.Y., U.S.A.

Received February 24, 1964

THE development of the eye pigments in insects has proven to be of advan- tage in studying the relation between gene-controlled processes and the appearance of morphological phenes. However, \vhile workers in this field have succeeded in the elucidation of metabolic pathways leading to the formation of the pigments and their dependence on the genetic constitution, our knowledge of the mechanisms controlling the timing of gene-controlled processes and of turning on and off the machinery provided by the suitable genetic constitution is far from being understood. In order to attack this problem, some experiments were undertaken \vith the hope to detect and time some of the steps in the ontogeny of the final phene.

The use of antimetabolites, in particular of halogenated pgrimidines, which during the past years have proved to he useful tools in the study of nucleic acid and protein biosynthesis, appeared to be a promising method for an attack on the problem of timing in development since their activity in metabolism is relatively well understood. Since it may be presumed that the initiation of a developmental process proceeds under rapid synthesis of enzymes and specific structures, antimetabolites might be expected to interfere with these processes in specific ways. In the formation of eye pigments in insects it is knoxvn that the pigments themselves are tryptophane-derived ommochrome pigments and pteridines, and that their deposition involves the formation of RNA containing precursor granules which show enzymatic activity [5, 11, 181. It was therefore decided to study the influence of 5-fluorouracil on developmental processes, since 5-fluorouracil may be expected to interfere with the synthesis of normal RNA.

1 This work has been supported by contract AT (30-l)-2902 of the U.S. Atomic Energy Com- mission.

z Present address: Institut ftir Entwicklungsphysiologie, Universitgit zu Kiiln.

Experimental Cell Research 37

Effect of 5-fluorouracil on eye pigmen tnry system

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:

1st duy: 6-12 and 1X-24 hr after pupation; %nd day: 30-36 and 42-48 hr after 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 6-12- hr-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 com- plications 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].


RESULTS

General effects of treatment.--Under the conditions employed, the pupal stage in Ephestia lasts 13 days. Only a few late-comers hatch on the 14th day. Owing to this insignificant variability it was sufficient to use only 5 pupae per group in the first control experiments. These animals were xvounded in the same region at difrerent times and sealed in the same way as the experimental pupae, without receiving a crystal of &FU. In contrast to the ohserva- tions made on growing larvae [lS, 161 the adults emerging from these wounded pupae hatched at the same time as uninjured controls, regardless of the time at which the wound was made. All developmental changes which were examined occurred at the same time in both groups.

Implantation of .5-FLJ affects these processes drastically. Implantation during the lst-3rd day causes almost immediate cessation of most visible developmental changes. Dissection on the 12th day reveals that the body c,avity is still filled with liquefied fatbody and tissue debris. No outgrowth of scales has taken place and the hypodermis has remained firmly attached to the overlying chitin. On the 6th and 7th day small brown spots appear within the still transparent wings and legs, the first indication of beginning degeneration. Nevertheless, the animals survive until at least the 13th day after pupation as indicated by their continued ability to move and by their respiratory activity.

A sudden change in response to 5-W treatment occurs between the 3rd and 4th day after pupation. Upon implantation on the 4th pupal day development continues normally up to the 11th pupal day, when the pigmentation of scales sets in. The pupa is able to develop into a complete, normally shaped moth with fully outgrown scales which can easily be pulled out of the pupal case. Its scales, however, are unpigmented. These animals are not able to hatch by themselves and implantation up to the 9th day still prevents hatching.

The distribution and spreading of the eye pigment.-The eye is the only organ that shows some continuing development even after implantation into 6-12-hr-old pupae. At this time the eye is colorless and transparent, so that the tracheae running through the head are clearly visible. Only in the dorsocaudal corner appears a faint orange tinge due to a few pigment spots (Fig. 1). During normal pupal development the pigmentation and differentiation process of the disc proceed from this region in concentric circles spreading over the eye 113, 171. The progress of pigmentation with increasing age of the pupa is shown in Fig. 1, upper row and in Fig. 2. The drawings in


Effect of A-fluorouracil on eye pigmentary system 57

66-72 I 78-M 102- 106 126 - 132 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.

Fig. 1 are based on the examination of 5 animals per class in the control group (upper row) and of 10 animals per class in the experimental group (lower row). In normal development, the pigment remains restricted during the first 3 days to a halfmoon-shaped region in the dorso-caudal area of the eye and consists of small red spots easily visible under the binocular. Its outer boundary is presented by a sharp bend in the surface of the eye.


58 11. illufh

Fig. 2.-Development of normal eye pigment in the Ephestia pupa. ((I) 42-48 hr; (h) 90-96 hr; (c) 126-132 hr; (d) 174-180 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 sub- sequently 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


Erect of 5-fluorourucil on eye pigmentury system 59

Fig. S.-Result of implantation of 5-F1J crystals into pupae of known ages. All observed at age of 10 days. (a) Implantation at 6-12 hr; (b) implantation at 18-24 hr; (c) implantation at 30-36 hr; (d) implantation at 54-60 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 depend- ing 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


60 W. Muth

the first day following treatment the pigment belt reaches the caudal edge of the eye and from the second day on a darkening of the originally orange pigment sets in, leading through bright and dark red to a brown color which remains unchanged after the 4th day. Implantation 12 hr later allows the pigment to trespass the sharp anterior boundary and in animals treated at 42-48 hr more than half of the eye becomes finally pigmented. Treatment with 5-FU at 66-72 hr does not stop the spreading process; the pigment reaches the anterior edge on the 5th day.

In all animals treated between 6 and 72 hr, the pigment finally present is of a brown color instead of black as in the controls and is concentrated in small spots distributed evenly over the pigmented zone. After the 8th day neighboring spots fuse, forming smaller or bigger masses of pigment, thus disturbing the original uniform pattern. Probably this is due to a lack in the normal histological differentiation. Though implantation into pupae 72 hr old does not stop the spreading of pigment before it reaches the edge of the eye, it is still capable of inhibiting the development of a homogeneous black coloration. However, eight out of ten animals treated 12 hr later showed the normal evenly black coloration on the 8th pupal day, while the other two had the brown spotted pigmentation characteristic for those implanted at 66-72 hr. From this time on 5-FU no longer interferes with the pigmentation potencies of the eye, although these are realized 3 to 4 days after the treatment.

Effects of 5-FU on the composition of the pigment.-The pigment of the adult Ephestia eye can be separated by paper chromatography into at least three components [13, 141: III, a yellow pigment with the highest R,-value; I, a carmine red pigment; and 0, a violet pigment closest to the starting line. A fourth component (II) has been described as present in very low amounts. All four components could be demonstrated in the material employed in these investigations, II as a very weak band. 0, I and II shared the characteristic reaction of ommochromes on being sprayed with diluted acetic acid solutions of KaSO, and NaNO, (red and yellow or brown coloration, respec- tively). The most rapidly migrating substance, III, however, which has been shown by Kuhn and Egelhaaf [14] to be xanthommatin gave no reaction with acidic NaSO, solution, and until this time attempts to extract it from our strain according to the methods described by Butenandt et al. [2, 31 were unsuccessful. Thus the question as to the nature of the com- pound in strain IVCR occupying the position of xanthommatin remains open. Spot 0 is constituted by ommin, and spot I by an ommatin of unknown constitution [ 131.


Effect of 5fluorouracil on eye pigmentnry system 61

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

TABLE I.

Component Normal day of Formation inhibited

appearance by 5-FU before day

III 2 0

I 4 n

0 7 4

large amounts one day later. Component II was omitted in this study, since its concentration is very low.

Eyes of animals treated with 5-FU during the lst-3rd pupal day and killed on the 10th day contained both components I and III but showed no trace of component 0. The amounts formed appeared to be reduced as compared to untreated eyes of the same age, probably in part due to the restriction of pigment carrying cells to limited regions of the eye (see Figs. 1 and 3). Even in animals implanted at 6-12 hr after pupation and killed on subsequent days, the occurrence of both pigments could be demonstrated at exactly the same age at which they appeared in the controls. The formation of component 0, on the other hand, was completely inhibited by implantation of S-FU up to 72 hr, but not by implantation into older pupae. Upon treatment at 78-84 hr the animals were capable of forming so much pigment, that it was possible to recognize all components by chromatography of the contents of single heads squashed on the starting line. Ten animals examined in this way on the 10th day contained a high amount of component 0, comparable to that found in the controls. Implantation into later stages also had no effect on the development of eye pigmentation, as far as could be judged by the appearance of the eye and by chromatography of the pigments. Table I summarizes the results of the chromatographic studies.

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


form two pigment components, spots I and III. As can be seen by comparing the upper and lower row in Fig. 1, the final pigmentation covers a larger area of the eye than it had reached at the time of implantation. Obviously the process blocked is not the pigmentation process itself but the spreading of a latent state prec.eding the deposition of pigment, which provides necessary conditions for the final synthesis of the pigment. This latent spreading process starts immediately after pupation and reaches the anterior edge at 72 hr. The pigmentation process itself, however, does not cxpantl from the dorso-caudal corner until 60 hr after pupation. Then it proceeds syn- chronously in both control and

Effect of 5fluorourncil on eye pigmentnry system 63

haaf [IA]. It can furthermore be concluded that 3-K does not affect the differentiation of the pigment itself, but a preparatory process which may be compared to determination. This process, in the case of the ommatins, spreads gradually over the eye in the course of 60 hr, preceding the dc- position of pigment. In the case of ommin, it seems to take place in all cells of the eye at the same time, about 3 days before the actual appearance of the pigment.

The \vay in \vhich 3-K affects the preparatory process for pigment formation is of interest, because it would permit some insight into the nature of the process. There remain, however, several different possibilities which cannot be distinguished on the basis of the present experiment. Since neither treatment with 5-bromouracil nor with uracil or fluorophenylalanine gave results comparable to the effects of 3-FU, it may he concluded that the effects are characteristic for this substance. As an analogue of uracil it may he expected to be incorporated preferentially into RXA, as has been she\\-n to he the case in bacteria [12], viruses [9] and mammalian cells [A]. But since insects show some peculiarities with respect to metabolic paih\vays (for example, review by Gilbert and Schneidermann, [S] it cannot be taken for granted that findings obtained with other groups of organisms can be applied to insects. If, however, this is assumed, two possibilities deserve consideration: S-176 might he incorporated into newly formed messenger KNA and iw activate it; or it might be incorporated into the HNA of the precursor granules, modifying their structure in such a way that the normal attachment of the enzymes active in pigment synthesis is disturbed, or that the enzymes themselves become abnormal [lo].

h second possible effect of 5-K to be kept in mind is its inhibitory action on DNA-synthesis upon its conversion into 5-fluorodeoxyuridine by the inhibition of thymidylate synthetase [7]. It may be supposed that this activity \vould play a prominent role in processes \vhich are accompanied by cell divisions. Since, according to Umbach [17] little cell division takes place in the eye after pupation, and the dividing cells are restricted to the distal layers of the eye disc which are poor in pigment-forming cells, this mechanism of 5-FU action on eye pigmentation appears more remote.

SUMMARY

The action of 5-fluorouracil on the development of the eye pigmentation in the pupa of Ephestia has been studied descriptively and by means of paper chromatography. It was observed that treatment with ;I-FU up to 60


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.

REFERENCES

1. ANDERS, G. and URSPRU~-G, H., Rev. Suisse Zool. 66, 259 (1959). 2. BUTENANDT, A. and NEUBERT, G., Z. Physiol. Chem. 301, 109 (1955). 3. BUTF,NANDT, A., SCHIEDT, U., BIEKERT, E. and KORNMAX, P., I.iebigs Ann. Chem. 586, 217

(1954). 4. CASPARI, E., Biof. Zenfrafbf. 74, 585 (1955). 5. CASPARI, E. and RICHARDS, J., Yearb. Carnegie Inst. Washington 47, 183 (1948). 6. CHAUDHURI, N. K., MONTAG, B. J., and HEIDELBERGER, C., Cancer Res. 18, 318 (1958). 7. COHEN, S. S., FLAKS, J. G., BARNER, M. D., LOEB, M. R. and LICHTESSTEIN, J., Proc. Naff

Acad. Sci. 44, 1004 (1958). 8. GILBERT, L. I. and SCHNEIDERMANN, II. A., Am. Zool. 1, 11 (1961). 9. GORDON, M. P. and STAEHELIN, M., Biochim. Biophys. Acfa 44, 458 (1959).

10. GROS, F. and NAONO, S., in R. J. C. HARRIS (ed.) Protein Synthesis p. 195. Acad. Press, New York, 1961.

11. HANSER, G., Z. Vererbungsl. 82, 74 (1948). 12. HOROWITZ, J. and CHARGAFF, E., Nature 184, 1213 (1959). 13. K~:HN, A., Rio/. Zenfralbl. 79, 385 (1960). 14. K~~HN, A. and EGELHAAF, A., Z. Vererbungsl. 90, 244 (1959). 15. MUTH, F. W., Wilhelm Roux Arch. Enfwicklungsmech. 153, 370 (1961). 16. POHLEY, H. J., Wilhelm Roux Arch. EnfwickZungsmech. 152, 183 (1960). 17. UMBACH, W., Z. Morphol. Oekol. Tiere 28, 561 (1934). 18. ZIEGLER, I. and JAENICKE, I>., Z. Vererbungsl. 90, 53 (1959).


the effect of 5-fluorouracil on the eye pigmentary system in ephestia kuhniealla

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