Studies on the utilization of dietary thymidine and thymine by Drosophila melanogaster larvae

0022-1910/80/1101-0775

J. Insect Physiol., Vol. 26, pp. 115 to 780. OPergamon Press Ltd. 1980. Printed in Great Britain.

$02.00/O

STUDIES ON THE UTILIZATION OF DIETARY THYMIDINE AND THYMINE BY DROSOPHILA MELANOGASTER LARVAE M. H. EL KOUNI* and DAVID NASH Department of Genetics, University of Alberta, Edmonton, Alberta. Canada T6G 2E9 (Received 14 December 1979; revised 29 June 1980)

Abstract-Deoxycytidine improves tolerance of Drosophila melanogaster to thymidine block, suggesting the presence of deoxycytidine kinase. At appropriate concentrations, a mixture of thymidine and deoxycytidine allows larvae to tolerate a higher concentration of 5-fluoro-2’-deoxyuridine than is tolerated with either thymidine or deoxycytidine alone. Thus, at this high concentration, 5-fluoro-2’-deoxyuridine appears to act primarily upon thymidylate synthetase, as it does at lower concentrations, rather than upon RNA metabolism, as has been suggested previously. Larvae can also be rescued from S-fluoro-2’-deoxyuridineinduced death by a high c_oncentration of thymine. The effect is enhanced by the presence of deoxyadenosine. Since this compound is known to increase the intracellular concentration of deoxyribose-l-phosphate, the main effect of thymine is probably due to its salvage utilization as a thymidine source. via the anabolic functioning of thymidine phosophorylase.

INTRODUCTION

activity of ribonucleotide reductase upon cytosine ribonucleotides. The result is the depletion of deoxycytidine triphosphate which can frequently be averted by the addition of deoxycytidine (MORRIS and FISCHER, 1963; MORRIS et al., 1963; WHITTLE, 1966;

SALVAGE utilization of thymidine and thymine was largely elucidated in cells and organisms blocked in de nave thymidylate biosynthesis (see CLEAVER, 1967; O’DONOVAN and NEUHARD, 1970). When appropriately defective mutants are unavailable, antimetabolites such as antifolate drugs and the deoxyuridine analogue 5-fluoro-2’-deoxyuridine (sub-

BUJERSELLand REICHARD, 1973; LOWE and GRINDEY, 1976; REYNOLDS et al., 1979). Deoxycytidine could

only be expected to relieve deoxycytidine triphosphate deficiency if deoxycytidine kinase activity were present sequently referred to as fluoro-deoxyuridine) can be (see Fig. 1). Under the conditions tested by EL KOUNI used to generate the ‘thymidineless’ state. and NASH (1974) deoxycytidine did not relieve the A definitive study of this kind has been performed toxicity of thymidine as might have been expected. on Drosophila melanogaster cells, using antifolate One interpretation of this finding is that there is no drugs (WYSS, 1977a). However, the fruit-fly is deoxycytidine kinase activity in Drosophila frequently used as a whole organism in experiments melanogaster, as suggested by BECKER (1976). involving nucleotide metabolism. Thus, it is of some However, PARKASH(1971) has shown that thymidine interest to observe the operation of the metabolic has a teratogenic effect upon Drosophila melanogaster system at the organismal level. EL KOUN~and NASH which can be suppressed by deoxycytidine. If these (1974), using a modified SANG’S (1956) defined phenomena are indeed analogous to thymidine medium and axenic conditions, studied the utilization toxicity and its reversal by deoxycytidine, it would of thymidine by developing larvae. Fluoroseem unlikely that BECKER’S(1976) observation deoxyuridine was used in their study, since antifolate represents a complete picture of the situation. drugs have a broader spectrum of effects than fluoroIn the present paper we report that it is possible to deoxyuridine, which primarily inhibits the enzyme in which deoxycytidine is devise conditions thymidylate synthetase. Fluoro-deoxyuridine inhibits antagonistic to thymidine toxicity and, hence, we development of Drosophila meLanogaster at 10e6 M conclude that Drosophila melanogaster larvae possess concentration and above (EL KOUNI and NASH, 1974). deoxycytidine kinase activity. In addition we Thymidine relieves this effect, reducing sensitivity by demonstrate the utilization of thymine in this system approximately two orders of magnitude, but is itself and present results which suggest that the primary toxic at high concentration (EL KOUNI and NASH, salvage pathway is via ‘reverse phosphorolysis’ by the 1977). enzyme thymidine phosphorylase. The toxicity of high concentrations of thymidine is thought to arise mainly from the increasing levels of MATERIALS AND METHODS intracellular thymidine triphosphate which inhibit the *Present address: Section of Biochemical Pharmacology, Division of Biology and Medicine, Box G, Brown University, Providence, RI 02912 U.S.A.

Germ-free Oregon-R females were allowed to oviposit for 24 hours on non-nutrient ‘egg-laying’ medium. Thirty newly-hatched, germ-free larvae were then transferred to shell-vials containing modified

175

M. H. EL KOUNI and DAVID NASH

116

dTTP

dCTP t dCDP*

Ribonucleotide reductose (2

I

t c

dTDP

P P

I

I dCMP

de nova - +dUMP

dTMP

synthetose

(I

)

t c

dTMP TdR

FdUMP kinase

dCMP CdR

TdR kinase

deominase

klnase

TdR t kinase

I

deaminase

mlase

‘ylose FU d R+- I - P

El

dR+-I-P

dR:l-P

1

Deoxyribomutose

dR-5-P

Fig. 1. The principle reactions involved in the interactions of deoxycytidine, thymidine, thymine and fluorodeoxyuridine. Three regulatory interactions are considered: (1) S-fluoro-2’-deoxyuridine monophosphate inhibits thymidylate synthetase; (2) thymidine triphosphate inhibits ribonucleotide reductase; and (3) deoxyribose-l-phosphate favors the anabolic function of thymidine phosphorylase. Nucleotides are represented by their constituent base (C, cytosine; T, thymine; U, uracil), prefixed by ‘d’ for 2’deoxyribosylated compounds. The level of 5’-phosphorylation is indicated with MP (monophosphate), DP (diphosphate) or TP (triphosphate). Deoxyribosides are represented in the form XdR. The prefix ‘F indicates fluorination at the 5-position. dR-1-P and dR-5-P are deoxyribose-land -5-phosphates, respectively. Compounds fed during the course of this study are shown in boxes.

SANG’S(1956) defined medium. Deoxyribonucleosides (thymidine, deoxyadenosine, deoxycytidine and fluoro-deoxyuridine) and thymine were incorporated into the medium at different concentrations. Additives, except fluoro-deoxyuridine, were all obtained from Sigma Chemical Co. (St. Louis, MO, USA) and were of reagent grade. Fluoro-deoxyuridine was a gift from Hoffman-LaRoche Ltd. At each concentration of the different supplements, replicate cultures derived from at least two batches of medium were used. (For full description of the Drosophila

Table

1. Effect of different concentrations pupariation’ among larvae grown

Molar concentration of thymidine

0

strain, components of media used, handling of larvae, and maintenance of axenic conditions, see EL KOUNI and NASH, 1974.) Pupariation and eclosion were monitored and recorded for each culture. RESULTS

Table 1 shows the interaction of thymidine (5 x 10m5-5 x lo-* M) and fluoro-deoxyuridine (10-7-10-3 M). Thymidine is clearly antagonistic to fluoro-deoxyuridine toxicity. It is not possible to

of thymidine on percentage on defined medium containing

Molar concentration 10-7 10-6

survival to eclosion fluoro-deoxyuridine

of fluoro-deoxyuridine 10-4 10-s

and

to

10-s

0

5 x 10-s (it) 5 x 10-4 5 x 10-s

(& (Z)

10-z (& 2.5 x IO-* (5:) 5 x 10-z (4” * Survival to pupariation is shown in parentheses minimum of 8 replicas of 30 larvae.

below survival to eclosion.

Each datum is based upon a

Studies on thymidine and thymine in D. melanogaszer

777

Table 2. Effect of different concentrations of deoxycytidine on percentage survival to eclosion and to pupariation* among larvae grown on defined medium containing fluoro-deoxyuridine Molar concentration of deoxycytidine

0

Molar concentration of fluoro-deoxyuridine lo-’ lo-6 10-s

0 (!:,

(;:)

2 (35)

0 (0)

(;:)

$87)

(3:)

(09

&

($

&

(:I

(::,

(::)

(;:,

(if,

(X)

(z:)

(::)

(;g)

10-4 0 (0) 0 (0) 0 (0)

5 x 10-s 5 x 10-4 5 x 10-a

(:I 0 (1)

to-2

* Survival to pupariation is shown in parentheses below survival to eclosion. Each datum is based upon a minimum of 6 replicas of 30 larvae.

Table 3. Effect of deoxycytidine (10m2 M) on percentage survival to eclosion and to pupariation* among larvae grown on defined medium containing fluoro-deoxyuridine with or without sub-lethal concentration of thymidine (2.5 x IOmZM) N ucleoside added

Molar concentration 10-7 10-e

0

of fluoro-deoxyuridine 10-r 10-4

10-a

None (ZR

(2:)

(3:)

(i)

(5:)

(5:)

(5:)

(5;)

(Z)

($

(GA)

(Zi)

(&

$87)

&

(z:,

(00) 0 (0)

Thymidine Deoxycytidine Thymidine and deoxycytidine _

(i) 2 (12)

(“0) 0 (0) 0 (0) (i)

* Survival to pupariation is shown in parentheses below survival to eclosion. Each datum is based upon a minimum of 8 replicas of 30 larvae.

Table 4. Effects of different concentrations of thymine on percentage survival to eclosion and to pupariation* among larvae grown on defined medium containing fluoro-deoxyuridine Molar concentration of thymine

0

Molar concentration of fluoro-deoxyuridine lo-6 10-s 10-1

10-4

0 (Z,

(&

(G,

(::)

(C)

(::)

(ii)

(&

5 x 10-s 5 x 10-a 5 X 10-s

(4:) 0.8 (53) 0.4 (35) 0.4 (55)

(i)

(0”)

(“0)

(“0)

(“0)

(“0)

(i)

(&

(

(202)

10-z (&

ffz,

* Survival to pupariation is shown in parentheses below survival to ecolsion. Each datum is based upon a minimum of 7 replicas of 30 larvae.

identify whether the limit to this effect is set by an intrinsic property of the response to fluorodeoxyuridine or by the toxicity of thymidine at 2.5 x 10-z M. Table 2 shows the interaction of deoxycytidine (5 x 1O-5-1O-2 M) and fluorodeoxyuridine (10-7-10-4 M). Comparison with equivalent concentration of thymidine (Table 1) shows

that, although it is never as effective as thymidine at optimal concentration (5 x 10e3 M), deoxycytidine produces a strong and consistent improvement in survival in the presence of fluoro-deoxyuridine. It should be noted that at concentrations above low2 M, deoxycytidine is itself somewhat toxic (EL KOUNI and NASH, 1977) and hence was not tested.

778

M. H.

EL KOUNI AND DAVID NASH

DISCUSSION

Table 3 shows the interaction of a sub-lethal dose of thymidine (2.5 x 10e2 M) and 10w2 M deoxycytidine in the presence of fluoro-deoxyuridine (10-7-10-3 M). Two major findings are evident: First, deoxycytidine counteracts mortality attributable to thymidine, although not completely. Second, the combination of thymidine and deoxycytidine allows survival of a small number of flies on 10m4 M fluorodeoxyuridine. This rather unimpressive datum is reinforced by the observation that 12% of the larvae pupariated and approx. 40% reached the late third instar stage (not shown in Table 3). In contrast, very few larvae survived beyond the first larval moult at 10 4 M fluoro-deoxyuridine in the presence of either deoxycytidine or thymidine alone. Table 4 shows the response of larvae grown on medium containing concentrations of fluorodeoxyuridine (10-7-10-4 M) to concentrations of thymine ranging from 5 x 10e5 to 10m2 M. Only high concentration of thymine ( 10e2 M) shows a significant effect on the toxicity of fluoro-deoxyuridine. Similar results were reported by GOLDSMITH and HARNLY ( 1950); only 8.3 x 10m3 M or higher concentrations of thymine were able to modify the toxicity of antifolate drugs on Drosophila. These results demonstrate the ability of the fly to utilize thymine to overcome the inhibition of de nova thymidylate biosynthesis exerted by fluoro-deoxyuridine. However, compared with utilization of thymidine (EL KOUNI and NASH. 1974 and Table 1), thymine is extremely inefficient in counteracting fluoro-deoxyuridine toxicity. Similar conclusions were derived from comparative studies on the utilization of the two compounds by a variety of systems (ZILBERSTEIN et a/., 1973; GOODMAN, 1974; SELMANand KAFATOS, 1974), including Drosophila cell cultures (Wuss, 1977a). The salvage utilization of thymine as a source of thymidylate is often limited by the availability of deoxyribose-l-phosphate. Thus the effect of deoxyadenosine as a source of deoxyribose-l-phosphate was studied. A concentration of 5 x 10m4 M was used, which avoids toxicity at higher concentrations (EL KOUNI and NASH, 1977). The results are shown in Table 5. Deoxyadenosine may even sensitize larvae to the presence of fluoro-deoxyuridine at 10m6 M and certainly does not improve survival. However, the same concentration of deoxyadenosine stimulates survival at 10m5 M fluoro-deoxyuridine provided IO-* M thymine is also present.

The toxicity of deoxycytidine

thymidine and its reduction

High concentrations of thymidine are known to inhibit growth ofcells in culture (MORRIS and FISCHER, 1963; MORRIS et al., 1963; WHITTLE, 1966; BLJJERSELL and REICHARD, 1973; LOWE and GRINDEY, 1976; REYNOLDS et al., 1979). The effect is generally considered to be the result of inhibition of ribonucleotide reductase by thymidine triphosphate. Animal ribonucleotide reductases act on all four diphosphorylated ribonucleosides (see HENDERSON and PATTERSON, 1973); however, substrate range is modified by the presence of specific inhibitors. In the case of thymidine triphosphate, the reductase activity towards cytidine diphosphate is reduced (MOORE and HURLBERT, 1966). Since the production of cytosine deoxyribonucleotides depends upon this activity (see Fig. 1), cytosine deoxyribonucleotide starvation ensues. In the presence of deoxycytidine kinase, deoxycytidine can reverse the thymidine block (MORRIS and FISHER, 1963; MORRIS et al., 1963; WHITTLE, 1966; BUJERSELL and REICHARD, 1973; LOWE and GRINDEY, 1976; REYNOLDS et al., 1979). Furthermore, the relatively slow uptake of deoxycytidine by animal cells can be stimulated upon addition of thymidine (PLAGEMANNet al., 1978). In our previous study with developing fruit-fly larvae, a preliminary attempt to mimic this effect of deoxycytidine failed (EL KOUNI and NASH, 1974). Subsequently, BECKER (1976) was unable to identify deoxycytidine kinase in Drosophila cell cultures, providing a rationale for this failure. However, given that BECKER (1978) has shown that several enzyme activities which originally were unidentifiable are, under specific nutritional conditions, present, the possibility remained that our negative results stemmed simply from the deoxycytidine concentration selected, rather than from an inherent lack of salvage utilization of deoxycytidine by deoxycytidine kinase. The reduction of thymidine toxicity by deoxycytidine demonstrated above is compatible with a conventional mechanism of thymidine block and suggests, therefore, that Drosophila larvae do possess deoxycytidine kinase activity. WYSS (1977b) has shown that deoxycytidine is antagonistic to the toxicity of deoxyguanosine in Drosophila cell cultures. Since deoxyguanosine

Table 5. Effect of deoxyadenosine (5 x IOmJ M) on percentage survival to eclosion and to pupariation* among

larvae grown on defined medium containing

Nucleoside added

Molar 0

fluoro-deoxyuridine

concentration 10-c

None

with or without

thymine

of fluoro-deoxyuridine 10-s

(10e2 M)

10-a

1 (49)

0 (0)

0 (0)

(107, 0 (0)

A

(s’:,

(2, 0 (37)

t:,

(;:,

(C,

&

A

(F!, Thymine (z:, Deoxyadenosine Thymine and deoxyadenosine

*Survival to pupariation is shown in parentheses minimum of 17 replicas of 30 larvae.

by

below survival

to eclosion.

Each datum

is based on a

Studies on thymidine and thymine in D. melanogastrr toxicity also stems from inhibition of ribonudeotide reductase and subsequent cytosine deoxyribonucleotide starvation which can be prevented by addition of deoxycytidine (LOWE and GRINDEY, 1976; CHAN, 1978; GUDAS et al., 1978; MITCHELL et al., 1978). WYSS’S(1977b) results can also be interpreted as suggesting the presence of deoxycytidine kinase activity in Drosophila. In addition since it is generally found that, in animals, deoxyguanosine is phosphorylated not by a specific kinase but by deoxycytidine kinase (DURHAM and IVES, 1970; IVES and DURHAM, 1970; ANDERSON, 1973; KRENITSKY et al., 1976; GUDAS et al., 1978), the high toxicity of deoxyguanosine to WYSS’S (1977b) cells and to Drosophila larvae (ELKOUNI and NASH, 1977) can also be argued to provide evidence for the presence of the enzyme. The interaction of thymidine. deox.vcvtidine andjluorodeoxvuridine At concentrations of fluoro-deoxyuridine of IO- 5 M and less, there is no evidence that the two naturally occurring deoxyribonucleosides interact in any way in reducing the toxicity of fluorodeoxyuridine. A conservative interpretation of the results simply suggests that thymidine block is still partially effective in killing larvae, despite the addition of IO-* M deoxycytidine. Thus, the survival of flies at these lower fluoro-deoxyuridine concentrations could be attributed to the presence of deoxycytidine. although this is not necessarily the case. M fluoroIn most circumstances, 10-4 deoxyuridine is exceedingly toxic to Drosophila melunogaster larvae. Thus survival of a total of five flies in the presence of deoxycytidine and thymidine, together with the high levels of partial development reported in the Results section, represents a very modification of the pattern of significant developmental response. This change has to be attributed to the combined presence of the two naturally occurring deoxyribonucleosides. In all probability, the result derives from the action of thymidine in overcoming a thymidine triphosphate induced by the action of fluorostarvation deoxyuridine on thymidylate synthetase. If this is the case, we must presume that, even at the higher concentration of fluoro-deoxyuridine ( 10m4 M), the predominant effect is still upon DNA synthesis, rather than upon RNA synthesis, as we have previously suggested (EL KOUNI and NASH, 1974).

77’)

(KAMMEN, 1967) required for reverse phosphorolysls (see HENDERSON and PATERSON, 1973; EL KOUNI. 1977). In our system the use of deoxyuridine was precluded by the use of fluoro-deoxyuridine to block thymidylats biosynthesis. However, in other systems purine deoxyribosides have been shown to be effective sources of deoxyribose-l-phosphate (KAMMEN, 1967; MUNCH-PETERSEN, 1967; BUDMAN and PARDEE. 1967; DALE and GREENBERG, 1972; GOODMAN, 1974). We therefore used deoxyadenosine to generate equivalent evidence. From the results shown in Table 5. \ce conclude that in ‘whole’ Drosophila larvae. as in cell culture systems, the inefficiency of thymine as a fluorudeoxyuridine antagonist is due to the limited of deoxyribose-l-phosphate. availability Hence thymine appears to be utilized via the anabolic function of thymidine phosphorylase. Indications of the presence of this enzyme in DrosopMu were reported by CLYNES and DUKE (1975). It is possible that deoxyadenosine alone may antagonise detoxification of fluoro-deoxyuridine, by elevating deoxyribose-l-phosphate levels. Deoxyribose-l-phosphate is a product of the reaction which converts fluoro-deoxyuridine to the much less toxic compound, S-fluorouracil. Thus, in the case of relief of fluoro-deoxyuridine toxicity with thymine, there must be a fairly subtle balance between the requirement for a deoxyribose-l-phosphate source (deoxyadenosine) and some means to remove deoxyribose-l-phosphate as a product of detoxification. However, it is not likely that the sole effect of thymine is removal of deoxyribose- Iphosphate; the demonstrated ability of thymidine to counteract fluoro-deoxyuridine toxicity (EL KOI;NI and NASH, 1974 and Table 1) cannot be explained in this manner, and suggests that any thymidine generated by reverse phosphorolysis of thymine would be used to circumvent fluoro-deoxyuridine block more directly. Furthermore, the superior effect of deoxyadenoseine and thymine (compared to thymine alone) would not be expected. None-the-less, in the final analysis, thymine probably does stimulate detoxification to some degree, as well as counteractmg the effect of fluoro-deoxyuridine by providing a thymidine source. AcX-now/rd~rmmt.F-This N.R.C. (Canada) Grant deoxyuridine was a gift Montreal, Quebec.

investigation was supported h) No. A3269 to D. N. Fluorufrom Hoffman-La Roche Ltd.

The utilization oj’ thymine

Utilization of thymine generally takes place through the combined action of thymidine phosphorylase and thymidine kinase. Since thymidine is utilized relatively efficiently it is clear that thymidine kinase does not limit thymine utilization. Thus, there remain only the possibilities that the uptake of thymine and the rate of reverse phosphorolysis (mediated by thymidine phosphoryiase) limit its utilization. WYSS (1977a) showed that the utilization of thymine is enhanced in the presence of deoxyuridine. We interpret this observation to indicate that in his cell system the anabolic action of thymidine phosphorylase is involved in the utilization of thymine, since deoxyuridine is a source of deoxyribose-l-phosphate

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