Insect Biochem., i973, 3, 4o9-417 • [Scientechnica (Publishers) Ltd.] 409

ON THE METABOLIC FATE OF ECDYSONE AND

3-DEHYDROECDYSONE IN CALLIPHORA VICINA

PETER KARLSON AND JAN KOOLMAN

Physiol.-Chem. Institut, Marburg, Deutschhausstr. 1-2, West Germany

(Received 2 February, I973)

ABSTRACT The metabolism of ecdysone in white prepupae of Calliphora vicina is

analysed. Ecdysone is hydroxylated to ecdysterone with a half-life of 2 hours. Both hormones are then converted into more polar products with a half-life of 12 hours. As a side reaction both ecdysone and ecdysterone are oxidized to 3-dehydroproducts. Reconversion of 3-dehydroecdysone to ecdysone occurs rapidly in vivo. The final products of ecdysone metabolism are conjugates which can be hydrolysed enzymatically with sulphatase and glucuronidase.

IN recent years several laboratories have become interested in the rate and the mech- anism of inactivation of ecdysone in various insects. The half-life of moulting hormones was measured in Sarcophaga (Ohtaki, Milkman, and Williams, 1968; Ohtaki andWilliams, 197o ) and inCalliphora(Karlson and Bode, 1969; Shaaya, 1969) and foundto be 2-6 hours, depending on the species and physiological stage of the insect. The titre and half-life of the hormones were determined with a bioassay responding not to ecdysone alone, but to the sum of all physiologically active substances present in the insect.

Since labelled ecdysone became available, the metabolic fate of this hormone has been studied in several insect species. Cherbas and Cherbas (197o) and Moriyama, Nakanishi, King, Okauchi, Siddall, and Hafferl (197o) found several metabolites after injection of labelled ecdysone; however, these products have not yet been identified. Heinrich and Hoffmeister (197o) described the occurrence of a glycoside of ecdysterone in Calliphora. From Calliphora 'white pupae', Karlson and Koolman (1973) isolated an enzyme that metabolizes both ecdysone and ecdysterone. The product of the reaction with ecdysone was identified as 3-dehydroecdysone (Karlson, Bugany, D6pp, and Hoyer, 1972 ). It was of interest to see whether this product of the enzymatic reaction is also one of the metabolites occurring in vivo, either as a transient product that is further metabolized, or as a final inactivation product. We therefore studied the metabolic fate of ecdysone and dehydroecdysone in 'white pupae' of Calliphora vicina.

MATERIALS AND METHODS MATERIALS

AU reagents were of analytical grade and were obtained from Merck (Darmstadt, West Germany).

INSECTS For all experiments, 'white pupae' of the blowfly Calliphora vicina R-D. (C. erythrocephala

Meigen) were used. The flies were reared as described by Karlson and Bode (1969).

41o KARLSON AND KOOLMAN Insect Biochem.

INJECTIONS

Injections of ecdysone (a-ecdysone) were made with an aqueous solution of the 23,24-3H2- labelled compound (gift of Schering AG, Berlin, West Germany) with specific activity 2"93 c. per re_mole. The volume injected per animal was 3 ~d., containing So ng. ecdysone with o'3 gc. radioactivity.

Injections of 3-dehydroecdysone were made with aqueous solution of 23,24-3H2-1abelled substance, which was prepared by incubating radioactive ecdysone with highly purified ecdysone oxidase, according to Karlson and Koolman (1973). An injection of 5 gl. of ioo ng. 3-dehydro- ecdysone (o.62 ~tc.; better than 85 per cent purity, TLC CHCIs/CH3OH, 8o : 2o) was given to each 'white pupa'.

The pupae were injected through the mouth and loss of labelled ecdysone was prevented by ligating ' white pupae' just behind the wound inflicted by the syringe needle. All injected animals were kept at 25 ° C. until homogenization.

EXTRACTION OF RADIOACTIVE MATERIAL

After varying periods of incubation (from 3o minutes up to 24 hours) samples of ten 'white pupae' were homogenized in I5 ml. methanol at S ° C. and centrifuged. The pellet was extracted for 15 minutes with I5 ml. boiling methanol and again centrifuged. Both supernatants were combined and brought to dryness under reduced pressure.

SEPARATION OF THE SUBSTANCES AND MEASUPa~MENT OF RADIOACTIVITY

The residue was dissolved in lOG ~I. methanol and freed from precipitates by centrifugation. The metabolites were separated by thin-layer chromatography (TLC). Plates (o'2s mm. thick) were prepared with silica gel HF~54 (Merck, Darmstadt, West Germany), and dried in air for 24 hours at room temperature. Chloroform/methanol (8o : 2o v/v) was used as the solvent system for chromatography. Unlabelled ecdysone (gift of Schering AG), ecdysterone (=2o- hydroxyecdysone, ~-ecdysone, crustecdysone; Rhoto-Pharmaceutical Co. Ltd., Osaka, Japan) and 3-dehydroecdysone (prepared as described by Karlson and others, 1972) were used as markers for identification of the radioactive compounds. The radioactivity was measured with a thin-layer scanner (Berthold, Wildbad, Germany) using a windowless methane flow counter. The unlabelled substances were located on the plates under u.v. light of 2S4 nm. wavelength.

RESULTS

The inactivation rate of ecdysone and its metabolites depends to a great extent on the physiological stage of the insects. All experiments described in this s tudy were carried out with early 'whi te pupae ' of Calliphora vicina. T h e early white pupae period was chosen, since the titre of moult ing hormones is high during this period (Shaaya and Karlson, I965a, b).

METABOLISM OF ECDYSONE

After injection of tri t ium-labelled ecdysone, the pupae were extracted and the metabolites separated by thin-layer chromatography (TLC). In these chromatograms the following labelled compounds (or classes of compounds) can be detected, ordered according to increasing R F:

Peak I, conjugates of ecdysone or its metabolites; peaks I I , I I I , IV, polar metabolites; p e a k ' p ' , ecdysterone; peak ' ~ ' , ecdysone; peak ' D ~ ' , 3-dehydroecdysterone; peak ' D~ ' , 3-dehydroecdysone.

T h e R~ value of the slowest migrating peak I corresponds to that of an ecdysone glucoside described by Heinrich and Hoffmeister (197o). But since in preliminary experiments substances of peak I are hydrolysed by a mixture of steroid sulphatase and ~-glucoronidase but not by a-glucosidase (Sannasi, i973), we suspect that ecdysones are conjugated with sulphuric or glucuronic acid rather than with glucose.

I973, 3 ECDYSONE METABOLISM IN Calliphora 411

The substances in peaks II, III, and IV are more polar than ecdysterone. The determination of the chemical structure is under way. Possibly they are hydroxylation

Oh

VZ k

lh

2k

6h

12 h

f6h

L,_ 24 h

f f l Z

FiG. 1.--Thin-layer radiochromatograms of Calliphora vlcina extracts after injection of tritium-labelled ecdysone into 'white pupae'. From top m bottom: increasing time of incubation; from left to right: direction of TLC. Discernible peaks of radioactivity were nmmed according to their increasing RF value: I, II , I I I , IV, ecdysterone (~), ecdysone (a) and 3-dehydroecdysterone (D~).

412 KARLSON AND KOOLMAN Insect Biochem.

products such as 2o,26-dihydroxyecdysone (Thompson, Kaplanis, Robbins, and Yama- moto, 1967). The dehydrocompounds (3-dehydroecdysone and 3-dehydroecdysterone) are less polar and run faster than ecdysone.

Fig. I gives the radiochromatograms of the metabolites, obtained at various times after injection of ecdysone. The first metabolite, appearing within half an hour, is ecdysterone. Radioactivity in peak I appears shortly afterwards, followed by peaks II-IV, with peak III the largest. The dehydroproducts can be detected as shoulders, but they are present only in small amounts (less than 3 per cent of the total radioactivity).

100

80

% of total radioactivity

60 f x

,~0

'~ ~ 12 16 20 2~ hours

FIG. 2.--Quantitative measurement of radioactive metabolites after injection of ecdysone into prepupae. Symbols: O, ecdysone; O, ecdysterone; A , substances of peak I I I ; ×, substances of peak I.

The time course of ecdysone disappearance and formation of the main metabolic pro- ducts during 24 hours after injection of ecdysone is shown in Fig. 2. Ecdysone is meta- bolized with a half-life of 2 hours. When its radioactivity decreases, that of ecdysterone increases, reaching its maximum at 8 hoursafter injection. The initial rise of ecdysterone titre accounts for 8o per cent of the initial loss of ecdysone. From these data we conclude that the main metabolic product of ecdysone in Calliphora white pupae is ecdysterone. The conversion of ecdysone into ecdysterone has been shown earlier by King and Siddall (i 969).

The radioactivity of ecdysone is then slowly shifted to more polar products, mainly those of peak III. The chemical nature of the substance(s) representing this peak is unknown; they are probably hydroxylation products of ecdysterone. Since the radio- activity of peak III reaches its maximum at 16 hours after injection and decreases there- after, this substance seems to be an intermediate in ecdysone metabolism.

1973, 3 ECDYSONE METABOLISM IN Calliphora 413

The metabolic end-products are conjugates appearing in peak I. The amount of radioactivity in peak I increases steadily for the first 24 hours and shows no decrease subsequently.

3-Dehydroecdysone, a metabolite described by Karlson and others (x972), may be a transient product appearing shortly after the injection of ecdysone. Similarly, some ecdysterone is oxidized to 3-dehydroecdysterone. Owing to the small amounts present the dehydroproducts have not been included in Fig. 2, but can be seen in Fig. i. Since they are less polar, they run faster than ecdysone. Metabolites with these properties have also been detected in other insects (Cherbas and Cherbas, 197o; Moriyama and others, 197o; Gorell, Gilbert, and Tash, 1972 ). It is of interest to see whether this product is further metabolized and in what direction.

METABOLISM OF 3-DEHYDROECDYSONE

In order to investigate the metabolic fate of 3-dehydroecdysone, we prepared this compound by incubating tritium-labelled ecdysone with purified ecdysone oxidase.

O h

Y ~ 16h

FIG. 3.--Thin-layer radioehromatography of Calliphora vidna extracts after injection of tritium-labelled 3-dehydroeedysone (Da) into 'white pupae'. Details as in Fig. I.

The obtained product was more than 85 per cent pure and was used for further experi- ments.

In contrast to the preceding experiment, no 22-hydroxylation of the injected substance yielding 3-dehydroecdysterone was observed. Instead, virtually all 3-dehydroecdysone is reduced to ecdysone within 3 ° minutes after injection (Fig. 3). This rapid reconversion of 3-dehydroecdysone to ecdysone may explain the rather small amount of 3-dehydro- ecdysone in the chromatograms of Fig. i.

It also explains that thin-layer chromatograms obtained after injection of 3-dehydro- ecdysone are very similar to those obtained previously by injecting ecdysone. Fig. 3

414 KARLSON AND KOOLMAN Insect Biochem.

shows some typical chromatograms of time o, 0. 5, 4, and i6 hours after injection of dehydroecdysone.

The time course of the metabolism of 3-dehydroecdysone is given in Fig. 4. Though very similar to Fig. z, there are some differences. The substance(s) of peak III are less

100 % of totol rodioactivity

80

, o 20

§ ;2 ~ = hoots FIO. 4.mQuantitative measurement of radioactive metabolites after injection of 3-

dehydroecdysone into' white pupae'. Symbols: 0, ecdysone ; O, ecdysterone; ×, substances of peak I.

prominent; thus it was not possible to include them in the figure. Moreover, the accumulation of conjugates is delayed.

DISCUSSION The experiments described above allow for the first time a quantitative evaluation of

the different metabolic pathways of ecdysone. The main metabolic routes are outlined in Fig. 5.

As indicated, ecdysone is metabolized rather rapidly with a half-life of 2 hours. The main product is ecdysterone, which is further metabolized with a half-life of approxi- mately 8 hours; it is converted at least in part to the more polar products of peak III and finally to conjugates (peak I). If both hormones are taken together, their half-life is approximately 12 hours.

This is in agreement with some earlier determinations of the biological half-life of the hormones measured by bioassays (cf. Rees, 1971); however, it should be kept in mind that the data given apply only to whole white pupae. The metabolism appears to be dependent on the species and physiological stage of the insects. This has been demon- strated in Locusta migratoria (orthoptera) by Hoffman, Koolman, Karlson, and Joly (1973 ).

I973, 3 ECDYSONE M~T~OLISM tN Calliphora 4x5

3-Dehydroecdysone, the product formed in vitro by ecdysone oxidase, occurs also in vivo as one of the metabolites, though only in minute amounts. Somewhat surprising was the finding that in vivo 3-dehydroecdysone is rapidly reduced to ecdysone.

The biological significance of these two reactions is not clear. At present the hypo- thesis cannot be ruled out that the oxidation of ecdysone to 3-dehydr°ecdys°ne is merely a side-reaction; if this is true, the reduction of 3-dehydroecdysone would be a protective mechanism against this reaction. Another possibility, to be discussed below, is that 3-dehydroecdysone is an active metabolite with a physiological function of its own.

The interconversion of ecdysone (ecdysone resp.) and its dehydroproduct should be catalysed by two different enzymes. Oxidation of ecdysone to 3-dehydroecdysone by

ecdy$one ~ 3-deh.-ecdysone

ecdysterone o_=..~. 3-deh-ecdysterone ~, , , J y -

substance(s), \ of peok ~ ~1

substances of peak I

FIG. 5.--Metabolic routes of ecdysone after injection into 'white pupae' of Calliphora.

ecdysone oxidase does not depend on any coenzyme (Karlson and Koolman, x973), the electron acceptor being molecular oxygen. The thermodynamic equilibrium of this reaction lies certainly far to the side of the dehydroproduct.

Therefore the rapid reduction of 3-dehydroecdysone must be due to another enzyme, 3-dehydroecdysone reductase. The electron donator has not yet been identified, but it may be NADH or NADPH.

With respect to the more polar products present in peaks II-IV, our findings corro- borate results of other groups (Cherbas and Cherbas, i97o; Moriyama and others, x97o ). Experiments to identify these compounds are under way.

Though the question of the nature of the highly polar metabolites of peak I remains to be answered, there seems to be little doubt that these compounds are the final inactiva- tion products of ecdysone and presumably also ecdysterone. This is strongly supported by the fact that they accumulate in 'white pupae' while the amount of all other meta- bolites decreases.

Normally, conjugates of this kind would be excreted through the Malpighian tubules. Indeed experiments carried out with Locusta showed us that this insect excretes injected ecdysone with the faeces, both in unchanged form and as conjugates, and that this excretion is stage-dependent (Hoffman and others, I973). Since the 'white pupae' used

416 KARLSON AND KOOLMAN Insect Biochem.

in this study are closed systems, the conjugates remain within the body, and it should be of interest to investigate in which tissue they accumulate.

As pointed out above, the main product formed from ecdysone is ecdysterone. This has already been observed by King and Siddall (i969) and interpreted by the assumption that ecdysone serves only as a precursor of ecdysterone, i.e., as 'prehormone' (King, x972).

However, recent results of Clever, Clever, Storbeck, and Young (i973) point to a special role of ecdysone and also allow one to speculate on a possible physiological func- tion of 3-dehydroecdysterone.

One of the earliest and most intriguing effects of ecdysone is the induction of puffs in salivary gland chromosomes (Clever and Karlson, I96O ). Working with isolated salivary glands, Clever and others (1973) have recently shown that the puff I-I8-C is specifically induced by ecdysone, while the locus IV-2-B responds to ecdysterone. However, in vivo (and presumably also in the cultured gland), ecdysone is rapidly converted to ecdysterone, resulting in a delayed response of the locus IV-2-B. After injection of ecdysterone in vivo, on the other hand, I-I8-C is also induced after a lag phase. This activity is attributed to a metabolite of ecdysterone.

A dehydroxylation of ecdysterone, yielding ecdysone, is highly unlikely. However, the results of Clever and others (1973) can easily be explained if we assume that the puff I-I8-C responds to 3-dehydroecdysterone (arising from ecdysterone) as well as to ecdysone or its dehydroproduct. Experiments to test this hypothesis are underway. In this context, it should be kept in mind that in the case of mammalian androgens it is not the testicular hormone, testosterone, but its metabolite, 5 (t-dihydrotestosterone, which is the active molecule (cf. Ofner, 1968 ).

SUMMARY The metabolic fate of ecdysone and 3-dehydroecdysone in Calliphora viclna has been

investigated by the use of tritium-labelled compounds and thin-layer radiochromato- graphy.

In 'white pupae' of Calliphora ecdysone is metabolized rapidly with a half-life of 2 hours. Thirty minutes after injection of ecdysone ecdysterone is detected. The amount of radioactivity of ecdysterone reaches its maximum 8 hours after injection. Ecdysterone is metabolized more slowly than ecdysone. It shows a half-life of about 8 hours. Both moulting hormones together show a half-life of 12 hours.

Ecdysone and ecdysterone are oxidized to the 3-dehydroproducts in only minute but detectable amounts (<3 per cent).

The group of ecdysones is converted into products of increasing polarity. Four sub- stances (or groups of substances) are discernible by thin-layer chromatography. Sub- stance(s) in peak III are only intermediate products of the ecdysone metabolism. Sub- stances of peak I (presumably conjugates) are final products of the reaction pattern.

In 'white pupae' of Calliphora 3-dehydroecdysone is converted into ecdysone within 3 ° minutes after injection.

ACKNOWLEDGEMENTS

We are obliged to Dr. Clever for sending us a preprint of his manuscript. The skilful technical assistance of Miss E. Kreilos is gratefully acknowledged. This study was supported by a Grant from Deutsche Forschungsgemeinschaft.

1973, 3 ECDYSONE METABOLISM IN Calliphora 417

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Key Word Index: Calliphora, ecdysone, metabolism of steroids, moulting hormones.