Sensitivity of insect ovarian tissues to various pteridines as tested in tissue culture

J. Insect Phyd., 1972,Vol. 18,pp. 1733to 1737.Pergamon Rest.

Printed in Great Britain

SENSITIVITY OF INSECT OVARIAN TISSUES TO VARIOUS PTERIDINES AS TESTED IN TISSUE CULTURE JANINA

SASKA,

BARBARA GRZELAKOWSKA-SZTABERT ZOFIA M. ZIELIl@KA

and

Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland

(Received 11 March 1971; revised 29 Fcbtumy 1972) Abtru%--The effect of pteridine derivatives and analoguea on the cell outgrowth from the ovarian explants of the waxmoth, Gall&z meUon&, was examined in hanging-drop cultures and cytochemical tats were made for auccinate and gluumed-phosphate dehydrogenaaea. Most of the derivatives and analoguea of 2-amine-4-hydrosypteridine injured to a lesser or greater extent insect ovarian tissues in vitro, depending on the drug structure and the concentration applied. Of all the pteridine derivatives and analogues tested only 2-amino-4 mercaptopteridine and its C-6,7-dimethyl derivative (1OpM) promoted cell outgrowth from the explants as did folate. INTRODUCTION

THE PROMOTING effect of folate and N-lo-formylfolate on cell outgrowth from the ovarian explants of the waxmoth and silkworm and the toxicity of 4-aminoanalogues of folate to insect ovarian explants and cells in culture have been previously shown ( ZIELIX~SKAand SASW, 1972). The authors interpret both phenomena in terms of a tetrahydrofolate requirement for one carbon transfer and utilization in insect metabolism. Some unconjugated pteridines have also been tested when added to the culture medium for the waxmoth. Since some of them affected the explants survival or cell migration, a number of pteridine derivatives and analogues were used in the examination of the sensitivity of the ovarian tissues of this insect in s&o. MATERIALS

AND

METHODS

Pharate adults (HINTON, 1971) of a waxmoth, GuZZerzkm&n& L., 4 or 5 days after larval-pupal ecdysis, were used as the source of ovarian tissues. Ovarian explants were cultured and their survival as well as the migration and multiplication of cells were checked as described previously (ZIELII~SKA and Ssm, 1972). The hanging-drop technique used enabled us to examine freshly dissected ovaries and cell outgrowths which would more probably respond to drugs in the same way as homologous tissues in the organism than would cells of an established cell line. The media used in this investigation were as follows: basal medium containing 10 per cent of heat-pretreated silkworm blood, salts, sugars, some organic acids, 1733

1734

JANINASASKA,BARBARA GRZELAKOWSKA-SZTAJNRT AND

ZOFIA

M. ZIELII&TSM

Difco lactalbumin acid hydrolysate as a source of amino acids, and Difco TC Yeastolate as a source of vitamins (JONESand CUNNINGHAM,1961; ZIEL~~SKA and SAXA, 1972). This basal medium was supplemented with folate or with one of the drugs under examination. Folate or pteridine and their derivatives or analogues were dissolved similarly as folate in O*1o/opotassium bicarbonate solution (if necessary with the addition of a few drops of 0.1 N KOH) and added to the basal medium at a final concentration of 10 a. Some of the drugs were also tested at 1 and O-2 PM concentrations. The ovarian explants were cultivated for a short time (4 days) in basal medium and in medium supplemented with folate or with one of the drugs. To eliminate the variations of the response of ovarian tissues from various insects, the explants from each of the individuals were cultured in a control medium and in an experimental one. In some of the experiments the ovarian tissues in the basal medium were cultivated for 3 days, and after changing the medium to one containing the drug, the culture was cont&med for two more days. All p&dine derivatives were synthesized and kindly supplied by Dr. K. Slavik, from the Charles University, Prague. RESULTS

AND DISCUSSION

Pteridines naturally occurring in insects (2-amine-4-hydroxypteridine, xanthopterine, isoxanthopterine, 6-carboxylatepteridine; ZIEGLER and H-EN, 1969) or their analogues of various types, namely compounds with additional aminomercapto- or methylsubstituents, were tested in the present study. 2-Amino_4_hydroxypteridine, which constitutes the pteridine fragment of folate, when present in the medium in a low concentration of O-2 PM, allowed the explants to survive moving and the cells to migrate abundantly (Table 1). When used as a 10 PM solution it did not have such a promoting effect; the explants survived, however, in the medium supplemented with this drug. They exhibited positive reactions typical for succinic and glucose&phosphate dehydrogenases. The positive test for glucose-6-phosphate dehydrogenase is a sign of the good viability of insect ovaries in culture as has been shown previously (ZIEL&XA and %%A, 1972). Both hydroxyderivatives of 2-amino_4_hydroxypteridine, namely xanthopterin (CA-OH) and isoxanthopterin (C-7-OH), in 10 PM concentrations killed the explants in a few hours. When diluted (1 or O-2 PM) these compounds did not always injure the explants since occasionally a few cells could migrate, but they became round and degenerated within 1 or 2 days. Of all the C-6- and C-6,7-substituted derivatives of 2-amino-4-hydroxypteriand carboxaldehyde-ones (O-2 and 10 PM) dine the methyl-, hydroxymethyl-, allowed the explants to survive moving with scant cell outgrowth. Somewhat more toxic seems to be the C-6,7-dimethyl derivative since no cell migration took place in the 10 PM solution. The 10 PM C-6-carboxylate and the cyclic C-6,7tetramethylene derivative rapidly killed the waxmoth ovarian tissues, whereas when more diluted (O-2 or 1 @I) they allowed some cells to migrate, but the

sENsrMvIn!

TABLE~-EFFE~~

OF INSECT OVARIAN

1735

TISSUES TO VARIOUS PTZRIDINXS

~F~~ME~~INEDEIRIVATI~E~ANDANAL~~~E~~N~~~~~.~~L~~A~~~AR~N TISSUE OF G. me&n&!,%a

G. m&m&a 0.2 /LM

Medium with the addition of None 2-Amino-&hydroxypteridine 2-Amino-4,6-dihydroxypteridine (xanthopterine) 2-Amine-4,7-dihydroxypteridine (isoxanthopterine) 2-&&o-4-hydroxy-6-methylpteridine 2-Amine-4-hydroxy-6-hydroxymethylpteridine 2-AminoA-hydroxy-6-carboxaldehydepteridine 2-Amino-4-hydroxy-6-carboxylatepteridine 2-Amino&hydroxy-6,7-dimethylpteridine 2-Amino-4-hy~o~-6,7-tetramethylenepteridine (cyclic) 2,4_Diaminopteridine 2,4,Diaminod-carboxaldehydepteridine 2,4,6-Triaminopteridine 2-Amine-4-mercaptopteridine 2-Amine-4-mercapto-6,7-dimethylpteridine 2-Amine-4-mercapto-6,7-tetramethylenepteridine (cyclic)

10 /LM Cell migration and outgrowth

Explants

Cell migration and outgrowth

Explants

Survived Survived Survived

Some Abundant None

survived Survived Dead

Some

Survived

None

Dead

None

Survived Survived Survived Survived survived survived

scant

survived

SomeSomeSomeSomescant-

Survived Survived Dead

scant scant scant-

scant scant scant

Survivd

Survived Survived Survived Survived Survived

Abundant

scant

Dead

Survived Dead Survived Survived

Some None

None None None

scantscantNone Abundant Abundant

Survived

Explants of the ovaries of the pharate adult 5 days after larval-pupal ecdysis were cultivated by the hanging-drop technique in basal medium with yeast extract as the sole source of vitamins, or in the same basal medium supplemented with one of the compounds listed above. For other details see Materials and Methods. Survived: Explants survived with active movements; positive reaction for succinic and glucose-6-phosphate dehydrogenases in all the types of ovarian cells. Dead: Explants became brown in 24 hr. Abundant: Abundant cell migration and after 2 days in culture formation of a large network of cells; positive reaction for both dehydrogenases. Some: Cell migration not very abundant, but formation of a fine network of cells; positive reaction for both dehydrogenases. Some-: Some cell migration, network of a few cells, glucosed-phosphate hardly detectable but with normal pattern. Scant : Scant cell migration, cells survived moving for a week or more without formation of cell network; negative reaction for glucose-6-phosphate dehydrogenase. Scant-: Scant cell migration, cells survived for not longer than 3 days; negative reaction for glucoseQ-phosphate dehydrogenase. None: No cell migration.

1736

JANINASASKA,BARBARA G RZELAKOWSIU-SZTABERT ANDZOPIAM. ~II~LI~~KA

normal pattern of glucose-6-phosphate dehydrogenase was observed only occasionally. All the 4-amino-analogties of 2-amino+hydroxypteridine tested proved to have some harmful effects on the ovarian explants and cells in vitro. Among them 2,4-diaminopteridiie and 2,4-diamino-6-carboxaldehyde seem to be less toxic than 2,4,6_triaminopteridine. Substitution of the pteridine ring in position 4 with a mercapto group seems to change also some biological properties of the compound. In contrast to 2-amino4-hydroxy derivatives the presence of 2-amine-4-mercaptopteridine and its C-6,7dimethyl derivative in the culture media was found to have a positive effect on the viability of the insect ovarian tissues in r&o, since it promoted abundant cell migration. The cyclic C-6,7-tetramethylene homologue of 4_mercaptopteridine, although it did not promote cell migration from the explant, seemed to be less toxic than its 4-hydroxy counterpart, because the ovarian explants survived in the culture media with active movements in tts presence. However, until now there has been no information regarding the participation of 4-mercaptopteridines in any of the known biological processes. It must also be added that 2-amino-4mercaptopteridine when applied as O-4 mM quickly killed explants. The sensitivity of insect ovarian tissues and cells in culture to some derivatives of 2-amino-4hydroxypteridine was rather unexpected as regards such pigment pteridines as xanthopterin and isoxanthopterin which are known to be synthesized and accumulated in some insects (WEYGANDet al., 1961; ZIEGLER and HARMSEN,1%9), and also in the case of 2-amine-4-hydroxy&zarboxylatepteridine which has often been identified in the pteridines isolated from insects (REMBOLD et al., 1964; I&RMGZN, 1966). However, the data concerning the amounts of pteridine derivatives present in insects cannot be converted to compare with those used in our media. Moreover, there are rather few data concerning the interaction of simple pteridines with folate related enzymes. Thus, 2-amino+hydroxy&carboxaldehyde and 2-amine-4-hydroxy-6-formylpteridine have been shown (ZAKRZEWSKI, 1960, 1963) to be cosubstrates for chicken liver folate reductase. For this enzyme such substituted simple pteridines as C-6-methyl-, 6-carboxylate-, and xanthopterin were found to be inert, whereas their C-6-formyl counterpart competitively inhibited the enzyme as the cosubstrate. The C-6-methyl derivative, inert to chicken liver folate reductase, has been shown to be a weak inhibitor of dihydrofolate reductase from Ehrlich ascites cells (BERTINOet al., 1965). 4-Amino anahave also been found to logues of C-6-hydroxy-, methyl-, or formylpteridines inhibit folate reductase from chicken liver (2 AKRZEWSKI,1963). Fifty per cent inhibition of dihydrofolate reductase from the waxmoth by several pteridines was achieved with O-4 mM C-6-carboxylate and C-6,7-dimethyl derivatives of 2-amine-4-hydroxypteridine and also with 2,4_diaminopteridine or with 0.1 mM 2,4,6-triaminopteridine (MANTEVPFEL-CYMBOROWSKA and GRZFUICOWSKA-SZTABERT, 1970). Thus, the dihydrofolate reductase of the waxmoth is inhibited in 50 per cent by these pteridines in concentrations about 40 times higher than those

SENSITIVITY OF INSECTOVARIANTISSUES TO VARIOUSmIDINEs

1737

negatively active in our biological assays for the sensitivity of the insect ovarian tissues to drugs. In conclusion, the toxicity of the 4-amino- analogues and of some of C-6- or C-6,7-substituted derivatives of 2-amino4hydroxypteridine for the insect ovarian tissues in Q&O,could not be explained in terms of interference with dihydrofolate reductase. No other pteridine analogue but 4-mercapto-derivative of 2-aminoA-hydroxypteridine, which in 0.4 mh4 concentration inhibited dihydrofolate reductase and was toxic to insect ovarian tissues in vitro, could promote cell outgrowth when applied in low concentrations. Acknowledgements-This investigation was supported in part by Grant No. FG-PO-214, Project No. E21-ENT-21 from the United States Department of Agricuhure, Foreign Agriculture Research Grants. The authors wish to thank Mrs. E. WALISZBWBKA and Miss E. WAci.AwFX for their technical assistance. REFERENCES

BERTWO J. P., PERKINSJ. P., and JOHNSD. G. (1965) Purification and properties of dihydro-

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