Cony. Biochem. Physiol. Vol. 79C, No. 2, pp. 435-439, 1984 Printed in Great Britam

0306-4492/84 $3.00 + 0.00 CI 1984 Pergamon Press Ltd

EFFECTS OF PURINE AMINO GROUPS ON THE

DEVELOPMENT OF DROSOPHILA

YEN-KUANG Ho,* DANIEL J. KoEHN,t RODNEY J. SOBIESKI,~ ANDREW J. CLIFFORDI and CAROLYN K. CLIFFORD:

*Department of Biological and Environmental Sciences, Morehead State University, Morehead, Keniucky 40351, USA.-Telephone: (606) 783-3376, tDivision of Biological Sciences, Emporia State University, Emporia, Kansas 66801, USA, IDepartment of Nutrition, University of California, Davis,

California 95616, USA

(Received 24 February 1984)

Abstract-l. The effect of a variety of 6-substituted purines on development of Drosophila melunogaster, eggs and larva, were studied.

2. Purine and 2,6-diaminopurine both were very toxic to egg development. 3. Adenine and 2,6_diaminopurine were moderately and equally toxic to larva development. 4. Substitution on the 6-position of the purine ring was very effective in regulating metamorphosis of

Drosophila.

INTRODUCTION

The influence of a variety of environmental chemicals

or mutagenic substances on genetic factors has been

studied (Vogel and Sobels, 1976). In addition, viruses,

bacteria, molds, higher plants, mice, hamsters, rats

and other organisms have been used as model systems to test many chemicals for their ability to induce mutations. Fruit flies, Drosophila melanogaster, are a commonly used model to evaluate mutagenesis or toxicity (Clark, 1982).

With respect to the biochemical mechanisms in- volved, dietary supplements of dimethylnitrosamine induced kidney tumors in rats by altering the methyl- ation on DNA at a number of different sites (Ayala and Kiger, 1980). Conditions that increase the fre- quency of deamination diminish the rate of the phage survivors. Dimethylation of adenine increases the resistance of Streptomyces erythraeus to erythro- mycin (Skinner and Cundliffe, 1983). Purine-base analogs, such as 2-aminopurine, and pyrimidine-base analogs, such as 5-bromouracil, were used to study T-even phage mutagenesis in replicating DNA (Freese, 1959). A purine analog has been shown to be an effective inhibitor of phosphoribosyl pyro- phosphate (PRPP) synthetase activity in cultured fibroblasts (Yen et al., 1981). Several pyrimidine and purine analogs are known to have different effects on the activity of PRPP synthetase in normal and leuke- mic blood cells (Danks and Scholar, 1982). Also, purine resistant mutants of Drosophila melanogaster were recently shown to be deficient in adenine phos- phoribosyl transferase (Johnson and Friedman, 1983) and PRPP is an essential cofactor for this enzyme. Mercaptopurine, an analog of adenine, is often used for treatment of acute leukemia because of its ability to interfere with nucleic acid biosynthesis (Angel, 1983).

Correspondence to: Dr. Yen-Kuang Ho, Department of Biological and Environmental Sciences, Morehead State University, Morehead, Kentucky 40351, USA.

Purine compounds are generally more toxic to Drosophila melanogaster than are pyrimidine com- pounds (El Kouni and Nash, 1977). Also Drosophila melunogaster responds differentially to media supple- mented with various nucleic acid bases (Vitt et al., 1982); guanine and cytosine reduced the number of offspring by 30% and 50”/ respectively, compared to controls while adenine and thymine completely pre- vented hatching. Recently, we (Ho et al., 1984) have shown that adenine at a concentration of 0.23% in the diet caused a 50% mortality among flies, whereas guanine at the same concentration caused only a 10% mortality.

Since little is known of the effects of purines with different configurations on Drosophilu melanogaster development, selected developmental stages, eggs and larva, of this organism were exposed to purine anal- ogs and studied. Although the purine analogs had similar heterocyclic ring structures, they had unique effects in different developmental stages of this fly. Specific locations of the amino and carboxyl groups on the purine ring were important determinants on the biological effects of these compounds.

MATERIALS AND METHODS

,The control medium (Ho et al., 1984) consisted of 37.5 g corn meal, 3.9 g agar-agar, 7 g yeast, 470 ml boiling water, 63 ml ethanol, 50 ml syrup, 47 ml malt extract, 2 ml zephiran 17x, 2 ml tegosept M and 1 g charcoal. Charcoal (0.15%) was added to the medium to facilitate visualizing the small white fly eggs against a dark background of the growth medium. Adenine, purine and 2,6-diaminopurine (Sigma Chemical Co., St. Louis, MO) were added to the control medium during preparation to provide a level of 0.23% in the medium. Approximately 5 ml growth medium was ad- ded to each 23 x 85 mm vial.

The study was performed in two sections. In one section, adult flies laid eggs directly in media containing chemicals and the development of eggs into larvae was evaluated. This group was called Group A larval testing. Also in this section, eggs deposited into control media were grown and

435

436 YEN-KUANG Ho et al.

the emerging larvae were harvested and seeded in media containing added purines and the development of these eggs to larvae was also monitored. This group is called Group B larval testing. In another section, only adult flies were fed the purines mixed in sucrose and then transferred to control medium to oviposit. The development of these eggs was monitored. This section is called adult testing.

Larval testing

Two pairs of virgin yellow-bodied D. melanogaster (first generation) from the Emporia State University, Emporia, KS stock culture collection were placed (to oviposit) in each of one control medium vial and in three experimental medium vials which contained adenine, purine or 2,6-diaminopurine. Each treatment was replicated four times. After 48 hr, parental flies were removed from the vials and discarded. The eggs were counted by using a dissecting microscope. These are referred to a Group A larval testing.

Seventy-two hours after removal of parentals (first gener- ation), the larva from one additional control medium vial were harvested and divided equally into vials containing no added purine, or containing added adenine, purine or 2,6-diaminopurine. Group A differed from Group B in that it was eggs from first generation flies (Group A) that were exposed to the purines while it was the larvae from first generation flies (Group B) that were exposed to the purines. Adults (second generation) which emerged from each vial of both groups were counted and saved for the adult testing section.

Adult testing

Briefly, adult testing, which was replicated four times, consisted of exposing second generation adult flies, har- vested from larval testing, to either sucrose (control) or to sucrose plus purine compounds for a single 48 hr period and then transferred to the control medium for an additional 48 hr to oviposit. To do this, one pair (one male and one female) of adult second generation flies from each of the vials in larval testing was placed in each vial containing Kleenex moisturized with 1.2ml aqueous solution of 10% sucrose as a control or 10% sucrose plus 0.23% adenine, purine or 2,6_diaminopurine for a 48 hr period. The flies were transferred to vials containing the control medium. Forty-eight hours later, parentals (second generation) were removed from the vials and the eggs were counted. Adults (third generation) that emerged were also counted.

RESULTS

Larval testing

Among the eggs from Group A that were exposed to purines, a concentration of 0.23% purine or 2,6-diaminopurine completely prevented metamor- phosis. Eggs in the control medium and the adenine- containing medium had a 83% and 53% survival rate, respectively (Table 1).

Among larvae from Group B that were exposed to chemical, purine at 0.23% was most damaging with only 9% survival of the exposed larva. Corresponding

Table 1. Larval testing toxicity from egg to adult

Group A’ No. of eggs No. of adults ‘A Survival

Control 31 k8 25 + 2 83 k 8 Adenine 36 k 3 19+_2 53 + 3 Purine 34 * 7 2*1 5*1 2,6-Diaminopurine 42 f 3 2*2 4&l

*Eggs were laid directly on experimental medium. The number of eggs, number of adults te emerge and percentage survival is reported.

Values are means i SEM.

Table 2. Larval testing toxicity from larva te adult

Group B’ No. of larvae No. of adults n/0 Survival

Control IOk I 9il 9oi 10 Adenine IlkI 7*1 64 k 9 Purine I1 i I I*1 9*9 2,6-Diaminopurine II i I 6k2 55* 17

*Larvae were harvested from control medium 72 hr after parental flies removed and then placed in experimental medium. The number of larvae per vial, number of adults to emerge and percentage survival is reported.

Values are means + SEM.

Table 3. Percentage death in fly developmental stages with different chemical exposure*

Egg-Larvae+Pupae+Adult “/, Death

Control ~ 7+-IO- 17 Adenine -ll+--364 47 Purine ~ 9+-91- 100 2,6-Diaminopurine -55+ -45- 100

‘All data summarized from Tables 1 and 2.

controls had a 90% survival rate whereas the larvae exposed to adenine and 2,6-diaminopurine had a 64% and 55% survival rate, respectively (Table 2).

The stages at which the flies die is summarized in Table 3 which is calculated from the data in Tables 1 and 2. In controls, 7% die between the egg to the larva stage and an additional 10% die between the larva to the adult stage. Similarly, 11% and 36% of flies given adenine die between the egg to larva and larva to adult stages, respectively. With purine, 91% of flies die in the larva to adult stage. Flies given 2,6-diaminopurine have the mortality at the egg to larva of 55% while in the larva to adult stages it is 45%. These data suggest that different purines arrest metamorphosis at different stages.

Adult testing

Adult second generation testing in which only flies were exposed to 10% sucrose had varying results of O-57% survival. Second generation flies in Groups A and B exposed to adenine during larval testing had a survival rate of 86% and Ox, respectively (Table 4). Second generation flies exposed to control diet during larval testing had 100% survival.

The adult testing control groups (Groups A vs B) had 100% survival while among flies exposed to adenine (Groups A vs B) had 86% and 0% survival, respectively (Table 4). The same was true for flies from the adult testing experimental group; that is, Group A all survived whereas only 17% of Group B survived (Table 5).

The numbers of eggs produced by second gener- ation flies fed 0.23% adenine are summarized in Table 6. Second generation adults, produced from eggs laid in media containing adenine (Group A) and fed with sucrose alone or sucrose + adenine produced 12 and 26% eggs, respectively. The larger percentage of eggs by adults fed sucrose + adenine suggests a higher resistance to adenine by this group. Second gener- ation adults produced from larvae grown in media containing adenine (Group B) and fed with sucrose alone or sucrose + adenine produced 67 and 32% eggs, respectively. The higher percentage of eggs on sucrose alone (67%) compared to sucrose + adenine (32%) is opposite of that seen in Group A.

Effects of purine groups on Drosophila 437

Table 4. Adult testing toxicity, control group*

No. of eggs No. of adults % Survival Group A B A B A B

Control 51* 5 37 k 3 57+4 37 * 2 100*4 lOOk2 Adenine 7*1 25 i 5 6+1 86 * 9

*The control group was exposed to 10% sucrose solution. Since the number of flies to emerge from purine and 2,6-diaminopurine containing medium was small, no adult testing was possible. Control and adenine flies were tested from both groups A and B.

Values are means + SEM.

Table 5. Adult testing toxicity, experimental group’

No. of eggs No. of adults % Survival Group A B A B A B

Adenine 15*3 l2&2 15+2 2*2 lOOf 17 f 16 2,6-Diaminopurine ~ 42 ? 2 6+1 - I4 f 3

*The experimental group was exposed to 10% sucrose and 0.23% chemical. Both Groups A and B for the adenine-treated flies were tested. Only Group B had enough flies for adult testing for 2,6-diaminopurine.

Values are means k SEM.

Table 6. Eee counts from adenine flies fed with 10% sucrose and 10% sucrose k 0.2% adenine

A B No. of No. oi

eggs % eggs %

Control 51 i 5’ lOOf 37 i 3’ 100+8 Adenine I* 1* 12*2 2.5 + 5’ 67i 14

(10% sucrose) Adenine 15*3x 26 f 5 12i2f 32 + 5

(10% sucrose + 0.2% adenine)

*Data were obtained from Table 4. tData were obtained from Table 5. Values are means 5 SEM.

The various purines are ranked in decreasing order of toxicity in Fig. 1. When all purines were included

medium (without charcoal). The average number of

at a fixed level (0.23%) purine was the most toxic flies to emerge for charcoal and regular medium is

while guanine and xanthine were least toxic. Adenine 40.0 f 17.5 and 24.3 f 5.5, respectively. Because of the overlap of standard deviation bars, mean values

and 2,6-diaminopurine were of intermediate toxicity. Figure 2 is a comparison of charcoal and regular

for both groups were not statistically different. There- fore, the charcoal had no deleterious effect.

r

0 PUrlne Ade

! 51 J”rln.3 Guonine Xonth ,ne controt

[ml Compound

Fig. 1. Order of toxicity. The sequence indicates toxicity of larva to adult. Percentage survival for larvae exposed to purine, adenine and 2,6-diaminopurine is 10, 50 and 55, respectively. Guanine and xanthine

have larva percentage survival rates of 90 and 93, respectively (Ho et al., 1983).

438 YEN-KUANG Ho et al.

CharcOOl Regular

1

!i mu Medsum

Fig. 2. Comparison of charcoal and regular medium. Num- ber of flies to emerge from charcoal and regular medium. Two pairs of parental flies per vial. Breeding time was 48 hr.

SE bars are shown.

DISCUSSION

Analysis of the larval testing results reveal at least two possible factors which may have contributed to the effects that purine amino groups have upon development of drosophila mefanoguster. One im- portant factor is the position of the amino group on the ring while the other factor concerns the solubility of the purines in aqueous media.

The data obtained from larval testing toxicity from larva to adult (Table 2) can be interpreted two ways. Firstly, the positions of amino groups on the purine ring may influence the percentage of surviving flies. Secondly, the more soluble a compound is, the easier it may enter the egg or larva and disturb biochemical life supporting mechanisms, hence, the observed difference may be related to the solubility of the test compounds.

Figure 1 illustrates the order of toxicity with regard to position of amino group. In general, the percentage survival rate for flies from purine, adenine and 2,6-diaminopurine can be considered 9, 64 and 55, respectively. Analysis of the data reveals that adenine (64%) and 2,6-diaminopurine (55%) have about the same effect upon larva to adult growth. Guanine at the same concentration allows 93% sur- vival for flies (Ho et al., 1984). It was concluded that the amino group in the number 6 position was more critical that the number 2 position. Both adenine and 2,6-diaminopurine have amino groups in the number 6 position, but adenine lacks the amino group in the number 2 position of the heterocyclic purine ring (Fig, I). If the number 6 amino group is removed and replaced with a carboxyl group (as in guanine, for example), the percentage survival increases to 937; (Fig. 1). The amino group in the number 2 position of guanine (2-amino-6-hydroxypurine) can be re- placed by a hydroxyl group, which converts guanine to xanthine (2,6_dihydroxypurine). This substitution did not increase the percentage fly survivability. Thus, the data demonstrate that the number 6 pos-

ition of the amino group was critical to survival from larva to adult.

Evaluation of data in Tables 1 and 2 in terms of percentages of death at the different developmental stages, shows that supplementation of 0.237: adenine in the medium was harmful to fly development from the larvae to the adults (Table 3). This is further confirmed by the day-to-day count of the populations from the egg stage through the larva, the pupa and to the adult stage (Banks and Ho, unpublished data). At the same concentration, purine is more detri- mental to larval stage during the fly metamorphosis, whereas 2,6-diaminopurine reduced by 55% (Table 3) the number of eggs which hatched to the larval stage. Analysis of the control and experimental data in Tables 4 and 5 shows that flies from Group A who developed from eggs and whose first generation par- ents were also exposed to adenine had tolerance to the chemical because 86 and 100% of these survived. Evidently, the egg exposed to the chemical allowed it to adapt to these conditions as it survived. Whereas, Group B flies from both control and experimental vials showed susceptibility to the adult treatment. Consequently, exposing eggs to chemical early in their development permitted only those who adjusted their biochemical processes to survive to adult stage. Thus, the purines had different selective pressures, depending on whether the egg or the larval stages were exposed to the chemical.

Two concerns with adult testing occurred during feeding on Kleenex. First, the concentrations of adenine and 2,6-d~aminopurine in solution were not the same due to their solubiiity differences. Adult flies exposed to the chemical may have ingested different amounts of each chemical. Secondly, the pHs of the three solutions tested were slightly different. Solu- tions of sucrose, adenine and 2,6-diaminopurine had pHs of 4.90, 5.53 and 5.05, respectively. However, due to the range of only 0.63 pH units and acidity of distilled water, pH probably was not a significant factor.

The adult flies were harvested from two sources: (a) the complete life cycle being developed in the adenine environment; and (b) exposure to adenine starting from the larval stage. From the egg count, the adults from (a) laid 12% eggs whereas the adults from (b) deposited 67% eggs (Table 6) compared to controls. The data may indicate less perturbation to the re- productive systems or mating behavior if the adult hatched from the egg that had not been exposed to the adenine.

Results from larval testing for egg to adult reveal (Table 1) that solubility of the chemical in question may be important to egg growth. Because purine is freely soluble in water, it is probably able to penetrate the egg membrane and disturb the biochemical mech- anisms. It is assumed that 2,6_diaminopurine is also soluble to an extent that will destroy the egg. During development from larva to adult, soiubility would also be a primary factor to consider. Because food consumption is very high during larval life, it was demonstrated that this stage is more sensitive than the adult stage (Clark, 1982). Further testing is necessary to establish the LD,, for purine and 2,6-diaminopurine before their effects on egg devel- opment can be explored.

Effects of purine groups on Drosophila 439

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