effects of 3β-(β,β-dimethylaminoethoxy)-steroids on sterol metabolism and development of bombyx...

Insect Biochem., 1976, Vol. 6, pp. 21 to 28. Pergamon Press. Printed in Great Britain

EFFECTS OF 3/~-(&/~-DIMETHYLAMINOETHOXY)-STEROIDS ON STEROL METABOLISM AND DEVELOPMENT OF BOMB Y X MORI*

H. HtKINO, Y. OHmuiI , T. SAITO, E. NAKAMURA, and T. TAKEMOTO

Pharmaceutical Institute, Tohoku University, Sendal, Japan

(Received 12June 1975)

Abstract--Three 3fl-(fl, fl-dimethylaminoethoxy)-substituted steroids, DAEA, DAEAM, and DAEC, have been found to be potent blockers of the enzyme system participating the conversion of fl-sitosterol into cholesterol in the silkworm larvae. Thus, a preliminary site of inhibitory action of these steroids is the terminal step in this conversion--from desmosterol to cholesterol, and a second- ary site of inhibitory action of DAEA and DAEAM is the initial step of this conversion--from fl-sitosterol to its immediate metabolite. These inhibitors have been proved to prevent larval development and metamorphosis of the silkworm that has been appreciably counteracted by administration of the moulting hormone.

INTRODUCTION

IT HAS been known that phytophagous and certain omnivoro us insects lack the ability for the de novo biosynthesis of the steroid skeleton and thus must receive their essential sterol from diet either in the form of cholesterol itself or a phytosterol which can be readily transformed to cholesterol. Transforma- tion of C~8 and C~9 phytosterols into cholesterol involves dealkylation of C-24 substituents whose mechanism is an important but little understood biochemical process. Although the conversion of fl-sitosterol into cholesterol has been shown to occur in the silkworm, Bombyx mori (IKEKAWA et al., 1966), a positively identified intermediate had not been reported when we initiated this work.

In an effort to elucidate the pathways of phytosterol metabolism in insects, hypocholesterolemic agents triparanol and 22,25-diazacholesterol which show inhibitive effects on sterol metabolism in a vertebrate rat were first demonstrated to be potent inhibitors of zfZ4-reductase leading to accumulation of desmosterol (24-dehydrocholesterol) as a metabolic intermediate in the conversion of fl-sitosterol to cholesterol in an insect, tobacco hornworm, Manduca sexta (SvoooDA and ROBBINS, 1967). Later, a number of steroidal hypocholesterolemic agents such as azasterols and 3fl-hydroxy-24- norhcol-5-en-23-oic acid were also revealed to accumulate desmosterol as a common metabolite of a number of different phytosterols, and cholesta- 5,22E,24-trien-3fl-ol as a metabolite of stigmasterol in several insects (SVOaOD^ and ROBBINS, 1968; SVOBODA et al., 1969; SVOBODA and RoBBINS, 1971). While it has been reported that another structural

* Steroid metabolism in Bombyx mori--III. This paper forms also Part XXVII in the series on Steroids.

type of compounds, 3B-(#,p-dialkylaminoethoxy)- substituted steroids, possess the hypocholesterolemic effect in rat by inhibiting the conversion of desmosterol to cholesterol (GORDON et al., 1961; CANTRALL et al., 1963). Our interest in the effects of these vertebrate hypocholesterolemic agents on the sterol metabolism, and growth and development of the silkworm has led to perform the present work. Although a number of potent analogs are known, we have chosen 3fl-(B, fl-dimethylaminoethoxy)-androst-5-en-17-one (DAEA) (GORDON et al., 1961) as a representative. 3B-(fl,B-Dimethyl- aminoethoxy)-androst- 5 -en- 17-one methyloxime (DAEAM) which is a possible active agent, and 3 p-(fl, B-diethylaminoethoxy)-cholest-5 -ene (DAEC) which is inactive in rat (CANTRALL et al., 1963) but may be expected to have an activity specifically in the insect, were also subjected to examination.

Part of the material contained herein has been published in a preliminary form (HIKINO et al., 1972).

MATERIALS AND METHODS

Preparation of materials DAEA and DAEC were prepared as reported

(GoRDoN et al., 1961; CANTRALL et al., 1963). DAEAM was prepared by treatment of DAEA with

methoxyl amine hydrochloride in pyridine under nitrogen at room temperature for 2 days followed by chrystallization of the resultant condensate from acetone as colorless needles, m.p. 73--75~C. NMR jcncts (ppm from internal TMS): 3H singlet at 0-90 (C~le)Hs), 3H singlet at 1-00 (C(I~)Hs) , 6H singlet at 2.44 ((CHs)sN-), 2H triplet at 2-70 (~ = 6, >N-CH~-CHg), 2H triplet at 3.66 (y = 6, -CH~-CHs-O-), 3H singlet at 3-78 (>N-O- CHa), 1H broad doublet at 5"30 (y = 4, Co)H). (Found: C, 73.95; H, 10.37; N, 7.13. CuH, oNaO2 requires: C, 74.18; H, 10"38; N, 7.21%).

21

22 H . HIKINO, Y. OHIZUMI, T . SAITO, E. NAKAMURA AND T. TAKEMOTO

Feeding of insects Table 2. T L C analysis of sterols from silkworm larvae

Fifth (last) instar larvae of Bombyx mori were reared R~ Value on Morus leaves coated with DAEA, DAEAM, or ste~ol DAEC at various concentrations. In the case of DAEA, Standard Silkworm administration through injection was also made as 50% Des~oste~oi 0,16 0.16 ethanol solution (agl/individual/day) using a micrometer- driven syringe every day while being fed on Morus Cholesterol 0.24 0.24 leaves. B -Sitosterol O, 24 O. 24

To examine the effects of DAEA and DAEAM on the metabolism of fl-sitosterol in larvae, larvae were reared on Morus leaves supplemented with DAEA or DAEAM and injected with [4-t4C]fl-sitosterol (0"4 gCi]3 gl olive oil/individual) at day 3.

To examine the effects of ]/-sitosterol and cholesterol of high concentrations on the sterol composition in larvae, larvae were daily reared from day 1 on leaves coated with the sterol (1% fresh weight of leaves) or on an artificial diet supplemented with ]/-sitosterol.

To evaluate the effects of DAEA on excretion of sterols from larvae, larvae were injected with [22,23-aH] ,8-sitosterol (5 IzCi/3 •g/2 #1 50% EtOH]individual) at day 1 and fed on Morus leaves added with DAEA there- after.

To evaluate the effects of ecdysterone on larvae treated with DAEA, DAEAM, and DAEC, larvae were fed until day 7 on leaves to which only a hypocholesterolemic agent was added, and then kept on leaves coated with both the agent and ecdysterone.

Analysis of sterols (1) Larvae were sacrificed at day 8 and extracted with

refluxing methanol (5 h × 3). Ether soluble portion of the methanol extract was saponified in 2N ethanolic potassium hydroxide at 70°C under nitrogen for 1 hr. The saponification products, after being extracted with ether, gave sterol preparation suitable for the following chromatographic analysis.

Gas-hquid chromatography (GLC) was carried out on two systems (cf., Table 1) on a Shimadzu model GC-1C instrument equipped with a flame ion detector using glass columns (350 cm) prepared by the method of HORNING et al. (1959) (Table 1). Identification of the

Table 1. GLC analysis of sterols from silkworm larvae

Relative retention time

Sterol Or-17 (0.75%) SE-30 (I.5%)

Standard Silkworm Standard Silkworm

Cholesterol 1.00 1.00 1.00 1.00

Desmosterol 1.17 1.16 1.07 1.08

fl-Sltosterol 1.57 1.57 1,48 1.48

Colmm ~Samx3S0cm; Column temp. 240°C

metabolite having the same retention time as desmosterol was corroborated by analysis of the G L C effluent using an LKB modal 9000 GC-MS instrument.

Th in layer chromatography (TLC) was performed on 50% silver nitrate-impregnated silica gel G T L C chromatoplates developed in chloroform-acetone (49 : 1) (Table 2).

(b) Larvae, which were raised on Morus leaves supplemented with DAEA or DAEAM, administered

Chromatoplates: Silica gel impregnated with 50% silver

nitrate; Developing solvent: chloroform-acetone (49:1)

with x4C-P-sitosterol at day 3, and sacrificed at day 6, were treated as above to afford sterol preparations. Each preparation was dissolved in acetone (0"2 ml) and an aliquot (4 to 20/A) was submitted to silica gel chromatography (¢ 7 × 20 mm). After development with hexane, the fraction eluted with benzene-ethyl acetate (5 : 1) was evaporated to dryness under reduced pressure in nitrogen, treated with heptafluorobutyryl imidazole (10/11) and ethyl acetate (5 gl) at room temperature for 10 min, and subjected to radio gas-liquid chromatography.

Radio gas-liquid chromatography was conducted on a Shimadzu model GC-4BM instrument equipped with a RID-2D (scintillation) detector using a glass column (100 cm) packed with OV-17 (1-5%) on Gas Chrom Q.

(c) Larvae, which received ~H-fl-sitosterol at day 1 and were sacrificed at day 8, and their combined faeces collected up to day 8 were separately extracted with refluxing methanol (5 hr × 3). Aliquots of respective methanol extracts were dissolved in Bray scintillator (I0 mI) and their radioactivities were determined by a Packard Tri -Carb liquid scintillation spectrometer model 3380 equipped with an absolute activity analyzer model 544.

Bioassay Standard test abdomens of the fleshfly, Sarcophaga

peregrina, were prepared as reported by OHTAKt et al. (1968). Mixtures of ecdysterone (graded doses) and DAEA (10 gg) in 50% ethanol solution (2/A/individual) were injected into each isolated larval abdomen by means of a microsyringe. The assays were evaluated 24 h after injection when each abdomen was scored as having undergone puparium formation according to K~LSON (1956).

Acute toxicity test in mice Acute toxicity test were conducted in adult male mice

of dd strain weighing approximately 20 g. DAEA, DAEAM, or DAEC was suspended in 3% gum arabic solution and the suspension (0.1 mI/10 g body weight) intraperitoneally injected and orally administered to mouse. Lethal criterion was done 48 hr after administration. The lethal effect (LD 5o) was determined by means of up and down method.

Histological examination After euthanesia, samples of the head, prothorax, and

silk gland were removed, fixed in Bouin fluid, washed with water, dehydrated with ethanol, embedded in paraffin, and sliced. The histological sections thus prepared were stained according to hematoxylin-eosin methods and subjected to microscopic studies.

Sterol metabolism and development of Bombyx mori

Table 3. Effects of ingested hypocholesterolernic agents on sterol composition of silkworm larvae reared on

Morus leaves

Agent (% wet weight) R e l a t i v e s t e r o l composi t ion (~)

B-Sitosterol Desmosterol Cholesterol

0 32 0 68

DAEA 0 . 0 0 0 0 7 5 35 22 45

0 . 0 0 0 7 5 34 35 31

0 , 0 0 7 5 43 28 29

0 . 0 2 5 50 21 29

0 . 0 7 5 55 15 30

0 . 1 2 5 48 7 55

0 . 1 7 5 48 0 52

0 . 2 5 40 0 60

DAE/LM 0 . 0 0 0 0 7 5 36 29 35

0 . 0 0 0 7 5 38 37 25

0 . 0 0 7 5 49 29 22

0 . 0 2 5 45 22 33

DAf/C 0 . 0 0 0 7 5 33 13 54

0 . 0 0 7 5 29 19 52 0 . 0 2 5 32 6 62

"Silkworms were reared on M0t'us l eaves coated wi th the sgents and

suerif tced at day 8.

RESULTS

Effects of DAEA, D A E A M , and D A E C on the sterol composition

Data in Table 3 demonstrate relationships between the dietary concentrations of the hypo- eholesterolemie steroids and the composition of the

23

Table 4. Effects of injected DAEA on sterol composition of silkworm larvae reared on Morus leaves

DAEA Relative sterol composition (g)

(pg/lndividual/day) 8-Sltosterol Desmosterol Cholesterol

30 34 17 49

100 42 8 50

500 47 0 53

Silkworms were closed wlth DAEA every day and sucrificed at day 8.

sterols isolated from the silkworm larvae. At the lower concentrations DAEA caused a significant accumulation of desmosterol, and reduced the amount of cholesterol. At the higher concentrations of DAEA, desmosterol did not accumulate, but instead, the relative quantity of p-sitosterol increased. DAEAM was somewhat more effective in blocking dealkylation than DAEA. DAEC was a much less effective inhibitor of sterol metabolism. Injection of DAEA was found to produce effects similar to those caused by its oral administration (Table 4).

Effects of DAEA and D A E . 4 M on the metabolism of p-sitosterol

Table 5 indicates the composition of the radio- active sterols in larvae which were fed on Morus leaves with DAEA or DAEAM and administered with l'C-p-sitosterol. On administration of D A E A

Table 5. Effects of hypocholesterolemic agents on metabolism of 14C-fl-sitosterol in silkworm larvae reared on Morus leaves

Agent (~ wet weight) Relative radioactivity (%)

B-Sitosterol Desmosterol Cholesterol

0 28 -- 72

DAEA 0 . 0 0 0 7 5 73 27 • - -

0 . 0 2 5 85 17 - -

DAEAM 0 . 0 0 0 7 5 71 29 - -

0 . 0 2 5 85 15 - -

Silkworms were injected with 1~C.~-sitosterol (5~g/indivldual)

at day 3 and sucrlficed at day 6.

Table 6. Effects of fl-sitosterol and cholesterol on sterol composition of silkworm larvae

Diet R e l a t i v e s t e r o l c o ~ p o s l t t o m (%)

B - S i t o s t e r o l Desmosterol C h o l e s t e r o l

Morus l e a v e s 32 0 68

Morus leaves+ 55 I 0 55 BLSitosterol

Morvs leaves+ 3 0 97 c h o l e s t e r o l

A r t i f i c i a l d ie t 7S 7 l g ( c o n t a i n i n g B-si tosterol )

S i l k w o r m s were reared ~a d i e t s c o n t a i n i n g the s t e r o l s and s u c r i f i c e d

at day 8.

24 H. HIKINO, Y. OHIZUMI, T. SAITO, E. NAKAMURA AND T. TAKEMOTO

c~

c.

~. 2 >, 13

_ "/

/ I ..roll / [ .~ ' " / 2 M'

• r ' ' / - " ' - "

.:~/~ ," A.~ ''~ - y

I I i i I I I I I 2 3 4 5 6 7 8

Feeding period, days

Fig. 1. Effects of DAEA on growth of the 5th instar larvae of the silkworm. Symbols: ," • control; • . . . . . • 0-000075%; 0--- . . . . • 0.00075%; • • 0.0075%; • . . . . . . . . • 0-025%;

• . . . . . • 0.125%.

and D A E A M , convers ion of desmosterol into cholesterol was s t r ic t ly inhib i ted , and the per- centages of the p recurso r fl-sitosterol and the in ter - media te desmosterol are dose d e p e n d e n t ; the h igher the dose of D A E A or D A E A M the h igher the con ten t of p-si tosterol and concur ren t ly the lower the con ten t of desmosterol .

3 o~

2 >,

o (]3

I 1 I i I I ] I 2 3 4 5 6 7 8

Feeding per iod, doys

Fig. 2. Effects of DAEAM on growth of the 5th instar larvae of the silkworm. Symbols: • • control; Q- . . . . . • 0-000075% ; 0--- . . . . • 0"00075% ;

• • 0-0075%; • . . . . . • 0-025%.

z

~ 2 >, 2~ o C~

4 --

/'I////°~ o //.

& A~A

i--'A'''A'- - -A ...A - - ~ "$1

I I I I I I I I I 2 3 4 5 6 7 8

Feeding period~ days

Fig. 3. Effects of DAEC on growth of the 5th instar larvae of the silkworm. Symbols : --~ • control; • . . . . . • 0-00075 % ; • • 0-0075% ;

• ......... • 0,025%.

Effects of fl-sitosterol and cholesterol on the sterol composition

In exper iments to verify a suppo t ion t ha t supp ly of a large a m o u n t of fl-sitosterol or cholesterol to Bombyx larvae m i g h t possibly lead to accumula t ion of an i n t e r m e d i a t e in the sterol metabol i sm, larvae were fed daily Morus leaves s u p p l e m e n t e d w i t h fl-sitosterol leading to the resul t t ha t desmostero l was indeed accumula ted in larvae (Tab le 6). Similar results were also ob t a ined in exper iments in w h i c h s i lkworm larvae were reared on an artificial d ie t conta in ing a large quan t i t y of fl-sitosterol (Tab le 6). Whi le admin i s t ra t ion of cholesterol na tura l ly raised the percentage of cholesterol in the total sterol f rac t ion in larvae bu t d id no t cause the accumula t ion of an in te rmedia te of the s terol me tabo l i sm (Tab le 6).

Effects of D A E A on the excretion of sterols

In the controls , fecal excret ion of labelled sterols after in ject ion of 8H-p-si tosterol was qui te slow, and abou t the hal f of the init ial dose was excreted in to

Table 7. Effects of DAEA on excretion of °H-fl- sitosterol in silkworm larvae reared on Morus leaves

Relative radioactivity (%) Agent (% wet weight)

Whole body Feces

0 45 SS

DAEA 0.00075 39 61

0.125 91 9 '

Silkworms were injected with 3H-~-sitosterol (3~g/individual) at day I

mnd sucrificod at day 8.

Sterol metabolism and development of Bombyx mori

Table 8. Effects of DAEA and ecdysterone on mounting and metamorphosis of silkworm larvae

DAEA Ecdys terone Mounting Pupa t ion F i n a l C h a r a c t e r

(% wet weight) ~ % day % day Larva Prothetelyk Pupa Adult

0 0 100 10.0 ± 0.3 100 14.4 ± 0.4 0 0 0 100

0 1.6 x 10 .6 100 9.0 ± 0.0 100 14.0 ± 0.3 0 0 0 100

0 1.6 x 10 -5 100 9.0 ± 0.0 100 13.7 ± 0.2 0 0 0 100

0.00075 0 100 10.3 ± 0.3 50 14.0 ± 0 .0 12 38 12 58

0.00075 1.6 x 10 -6 100 9 .4 ± 0 .2 100 14.5 ± 0 .0 0 0 0 100

0.0075 0 100 11 .1± 0.2 38 15.3 ± 0 . 3 0 62 0 38

0.0075 1.6 × 10 -6 100 9.0 ± 0.0 I00 14.8 ± 0 .3 0 0 11 89

0.075 0 , 63 14.8 ± 0 .4 25 20.0 ± 0 .0 25 50 25 0

0.075 1.6 x 10 -6 88 12.1 ± 0 .2 25 17.0 ± 0.0 25 50 20 0

0.075 1.6 x 10 -5 88 9.9 ± 0.1 37 15.0 ± 0 .0 25 38 25 12

0.125 0 0 0 -- 75 25 0 0

25

Silkworms were reared on Morus. leaves containing DAEA and orally administered with ecdysterone

a t day 8; 8 l a rvae used i n each expe r imen t

Prothetely and metathetely

feces during 7 days period (Table 7). DAEA at the low concentration gave a little effect on the rate of excretion of 8H-/~-sitosterol and any of its metabol- ites, while DAEA at the high concentration caused severe reduction of the excretion rate (Table 7).

Effects of DAEA, DAEAM, and DAEC on growth The effects of the dietary-administered hypo-

eholesterolemic steroids on growth of the larvae (as measured by increase in weight) are shown in Figs. 1 to 3. At the lower concentrations, DAEA caused less retardation of larval growth; but at concentrations of 0.025% or greater, DAEA caused more severe retardation of growth. Likewise, D A E A M had no appreciable effects on larval growth at the lower concentrations but effeeted larval growth at concentrations of 0.025%. Interestingly, DAEC was the most potent inhibitor of growth and when larvae were fed on the Morus leaves supplemented with DAEC even at relatively low concentrations, all of them died before they reached the mature stage.

Effects of DAEA, DAEAM, and DAEC, and~or ecdysterone on mounting and metamorphosis

Tables 8-10 summarize the inhibitory effects of the dietary-administered hypocholesterolemic agents on development and the recovering effects of the moulting hormone on the degenerated insects. When DAEA or D A E A M at concentrations of up to 0.0075% was administered, some insects experi- enced a few days delay of mounting and larval- pupal and pupal-adul t apolyses; the remaining larvae, some of which showed the characters of prothetely, were unable to spin cocoons. At the highest concentration (0.125%), the larvae experi- enced difficulty in spinning, and those which did apolyse developed into larval-pupal intermediates.

When larvae were reared on Morus leaves containing DAEA, DAEAM, and DAEC, and ecdysterone was administered at day 8, the retardation of growth, mounting, and metamorphosis of the insects caused by these inhibitors is appreciably counteracted by administration of the moulting hormone.

Table 9. Effects of DAEAM and ecdysterone on mounting and metamorphosis of silkworm larvae

DAEAM Ecdysterone Mounting Pupation Final character

(% wet weight)~ % Day % Day Larvae Prothetely ~ Pupa Adult

0 0 100 10 100 14 0 0 0 100

0 1.6 × 10 -6 100 10 100 14 12 0 0 88

0.00075 0 88 11 58 16 12 50 0 38

0.00075 1.6 x 10 .6 76 10 76 16 25 0 38 58

0.0075 0 25 13 0 -- 76 24 0 0

0.0075 1.6 × 10 .6 88 10 38 15 12 50 0 58

0.025 0 12 13 0 -- 80 12 0 0

0.025 1.6 × 10 -6 12 13 0 -- 88 12 0 0

~$£1kworm were r e a r e d on ~orus l eaves c o n t a i n i n g DAE/~4 and o r a l l y a ~ i n i s t e r e d w i t h ecdysterone a t day 8; 8 l a r v a e used i n each e x p e r i m e n t

b p r o ~ h e t e l y a n d ~ e t a t h e t e l y

26 H. HIKINO, Y. OHIZUMI, T. SAITO, E. NAKAMURA AND T. TAKEMOTO

Table 10. Effects of DAEC and ecdysterone on mounting and metamorphosis of silkworm larvae

DAEC Ecdysterone

(% wet weight )~

Mounting Pupation Final character

% Day % Day Larvae Prothetely ~ Pupa Adult

O 0 I00 i0 I00 14 0 0 0 I00

0 1.6 X I0 -6 I00 I0 I00 14 12 0 0 88

0.00075 0 63 10 63 1S 12 25 0 65

0.00075 ] .6 × i0 -6 76 I0 76 15 12 12 0 78

0.0075 0 [) -- 0 100 0 0 0

0.0075 1.6 x i0 -6 0 -- 0 -- I00 0 0 (}

0.025 0 0 -- 0 -- i00 0 0 0

0.025 ] ,6 x 10 -6 0 -- 0 -- I00 0 0 0

Silkworms were reared on Morus leaves conta in ing DAEC and o r a l l y adminis tered with

ecdysterone a t day 8; 8 lervae used in each experiment

P r o t h e t e l y and metathetely

The silk glands of the hypocholesterolemic agent-induced abnormal larvae were normal in appearance. Histological examinations of the insects in prothetely and metathetely showed no appreciable changes in brains, corpora allata, prothoracic glands, and silk glands.

Effects of DAEA on the moulting hormone activity of ecdysterone in the Sarcophaga test

DAEA, DAEAM, and DAEC (5 ttg/individual) when injected in combination with graded doses of ecdysterone gave no effects on the dose response curve of the moulting hormone in the Sarcophage test.

Acute toxicity of DAEA, DAEAM, and DAEC in mice

The LD~0 values of the hypocholesterolemic steroids determined in mice by means of up-and- down method are tabulated in Table I1. Main symptoms induced by the steroids are clonic con- vulsion for DAEA and DAEAM, and stretching for DAEC.

Table 11. Acute toxicity of hypocholesterolemic agents in mice

Agent LD~ (mg/kg)

i , p . p .o .

DAEA 39 404

DAEAM 25~ 752

DAEC >3000 >3000

LDs0 values were determined by up and down method

in 48 hours mortality response

DISCUSSION

In order to elucidate the pathway of the metabolism from B-sitosterol to cholesterol in Bombyx mori larvae, three synthetic steroids DAEA, DAEAM,

and DAEC were administered if accumulation of some metabolic intermediates occurs. As is evident from Table 3, each steroid at the lower concentrations caused a significant accumulation of desmosterol which is not found in normal larvae, and diminished the percentage of cholesterol in a total sterol fraction, indicating that in the silkworm larvae the metabolism of fl-sitosterol to cholesterol proceeds through the intermediate desmosterol. The order of the inhibitory effect against the A *4- reductase was as follows: D A E A M > D A E A ~ DAEC, and the activity of DAEC was estimated to be less than 1/I00 of that of DAEAM. This order is that also observed in the hypocholesterolemic activity in rats. Thus, 3fl-(fl,fl-diethylaminoethoxy)- androst-5-en-17-one methyloxime (an analog of DAEAM) is more than 20 times as active as DAEA, and DAEC shows no hypocholesterolemic activity in rat. It is interesting to note that DAEA exhibits the activity in both the insect and vertebrate, while DAEC shows the activity only in the insect though the effect is weak.

Upon raising the concentrations of DAEA and DAEAM, accumulation of desmosterol rather decreased, and instead, the relative quantity of p-sitosterol increased (Table 3), suggesting a more complete interference with the dealkylation mechanism and blockage of the conversion of fl-sitosterol into its immediate catabolite. In confirmation of this conclusion, more direct evidence was obtained by the tracer experiments. Thus, administration of DAEA or DAEAM inhibited the transformation of fl-sitosterol to its immediate catabolite resulting in the retention of the ingested sterol fl-sitosterol and the higher the dose of the hypocholesterolemic steroid administered the high the percentage in the total sterol portion (Table 5). These steroids thus constitute the first example inhibiting the enzyme system which conducts the conversion of fl-sitosterol into desmosterol. While DAEC did not exhibit such a double-faced effect. Thus, even at the higher concentration it specifically inhibited the transformation of desmosterol to cholesterol and

Sterol metabolism and development of Bombyx mori 27

did not bring about the accumulation of ]~-sitosterol (Table 3).

Further increase of the DAEA concentration up to 0.175 and 0.25%, the percentage of B-sitosterol in the sterol fraction decreased while that of cholesterol increased (Table 3), and thus the sterol composition approached that of the normal larvae. This observation may be considered to be due to the virtual suspension of the ingestion, metabolism, and excretion of fl-sitosterol in the larvae as judged from the facts that the body weight gain of the larvae which received such high concentrations of DAEA was not found from day 1 of the 5th instar when the feeding experiment started (Fig. 1) and that the sterol excretion from the larvae was remarkably depressed by administration of a higher amount of DAEA (Table 7).

Accumulation of desmosterol observed in larvae which were fed on Morus leaves coated with B-sitosterol or artificial diet containing B-sitosterol supports that desmosterol is an intermediate of the sterol metabolism and further suggests that the conversion of desmostcrol into cholesterol is the rate- determining step in this dealk~dation reaction system. Thus, it is highly probable that the rise of the substrate concentration caused by absorption of B-sitosterol accelerates the each reaction but the reduction of desmosterol to cholesterol proceeded most slowly so that the intermediate desmosterol was accumulated.

In the present multienzyme sequence catalyzing the conversion of B-sitosterol to cholesterol, it might be expected that a high level of cholesterol inhibit some enzyme in the system, leading to accumulation of an intermediate. In fact, however, cholesterol dosed with Morus leaves as well as desmosterol accumulated by administration of DAEA and D A E A M did not bring about accumulation of another intermediate.

The fact that the accumulation of desmosterol reached up to 37% in the total sterol fraction and exceeded that of cholsterol in certain conditions (Table 3) demonstrates that if the total sterol content in larvae does not significantly vary, the greater part of the accumulated cholesterol has been biosynthesized from p-sitosterol during the period of the 5th instar. Further, it was shown that :4C-fl- sitosterol administered into normal larvae at day 3 of the 5th instar was largely converted into cholesterol in 3 days (Table 5). These findings reveal that the conversion of fl-sitosterol into cholesterol is efficiently mediated in the 5th instar larvae of the silkworm.

The hypoeholesterolemic agents caused the inhibition of growth and development of the silkworms; the higher the dose administered the severe the inhibition. Inter alia, DAEC exhibited a far more intense toxicity against the insect to interfere the growth than DAEA and D A E A M (Figs. 1, 2, 3). SVOBODA et al. (1967) previously postulated that

retardation of larval growth might be due to the effect of desmosterol or some other sterols accumulated on the basis of their feeding experiments on the tobacco hornworm with triparanol and 22,25- diazacholesterol. However, during the course of the present study on the effects of dietary-administered DAEA, DAEAM, and DAEC on growth of the silkworm larvae, these steroids at the lower concentrations where significant accumulation of desmosterol was found caused less retardation of larval growth; but at higher concentrations where desmosterol did not accumulate, DAEA caused more severe retardation of growth (Table 3, Figs. 1 to 3). Furthermore, DAEC, which has the least inhibitory effect against the enzymatic reduction of desmosterol to cholesterol, showed the strongest inhibition of the larval growth (see above). These observations therefore indicate that there is no direct relationship between accumulation of desmosterol and inhibition of growth. I t may be worthy to note that, contrary to the toxicity against the insect, DAEC showed a much lower toxicity than DAEA and D A E A M against the vertebrate mouse (Table 11). The inactivity of DAEC in rat was previously assumed to be due to low absorption of the agent by the oral route of administration (CANTRALL et al., 1963). However, this supposition was found to be unlikely by the fact that the acute toxicity of DAEC is very low in mice in both oral and intraperitoneal administrations. Therefore, the inactivity of DAEC in the vertebrate is considered to be due to its property in situ. No persuasive explanation about tile discrepancy in activities of DAEC in the insect and vertebrate could be presently made.

Possible explanations of the inhibitory action of the agents, DAEA, DAEAM, and DAEC, on growth and development of the silkworm are: (1) the agents may affect physiological systems other than sterol metabolism; (2) they may act as antagon- ists of moulting hormones, or (3) they may also block the conversion of cholesterol into moulting hormones, resulting in a shortage of the latter which are essential to growth and metamorphosis. I t has been observed that in rarely occurring non-cocoon larvae and larvae in prothetely, the middle portions of the silk glands are degenerated which is considered to be due to the hormonal unbalance caused by the functional disorder of the corpora allata (FUKUDA, 1956). Contrary to what is found in the above abnormal larvae, in non-cocoon larvae and insects in prothetely induced by DAEA and D A E A M the silk glands appeared normal, so that the mechanism for the steroid-induced abnormality is different from that for the spontaneously formed one. The finding that histological examinations of the larvae in protlletely and metathetely induced by DAEA and D A E A M exhibited no changes in the brains, corpora allata, and prothoracic glands which play important roles in metamorphosis of insects demonstrates that the abnormality is not brought

28 H. HIKINO, Y. OHIZUMI, T. SAITO, E. NAKAMURA, AND T. TAKEMOTO

about by morphological origin. PRUVHI (1927) previously described that under the influence of unsuitable temperatures and a gas given out by the beetles the prothetelic changes in certain insects were observed which was considered to be the result of the inhibited metamorphosis rather than acceler- ated development. In the larvae in prothetely and metathetely induced by the administration of DAEA and DAEAM, the larval stage was prolonged, suggesting that presently observed abnormality is similar to that found previously. These agents thus provide one of the rare examples where the prothetely is caused by chemicals. The hypocholesterolemic agents were examined in terms of their ability to antagonize the moulting hormone ecdysterone, which induces puparium formation of isolated larval abdomens of the flesh fly, Sarcophaga peregrina. It was found that the agents have no such an antagon- istic activity, excluding the second possibility. The observation that the retardation of growth, apolysis, and metamorphosis of the insects caused by DAEA, DAEAM, and DAEC was appreciably counteracted by administration of the moulting hormone (Table 8, 9, 10), suggests that the agents cause lack of moulting hormones due to an additional block in the metabolic pathway from cholesterol to moulting hormones.

While the present work has been in progress, MORISAKI et al. (1972) demonstrated by incorpora- tion and trapping experiments that fucosterol and fucosterol 24,28-epoxide are probable intermediates in the conversion of fl-sitosterol into cholesterol in the silkworm. Accumulated data have thus clarified a dealkylation route, /~-sitosterol -+ fucosterol -+ fucosterol 24,28-epoxide -~ desmosterol -~ cholesterol, in the silkworm, B. mori.

Achnozoledgements--We are obliged to Dr. H. Morn, Teikoku Hormone Mfg. Co., Ltd., for the gift of 5- dehydroepiandrosterone, to Dr. J. N. KAPLANIS, U.S. Department of Agriculture, for carrying out the GC-MS determination, to Mr. H. Miyazaki, Nippon Kayaku Co., for performing the radio gas-liquid chromatography, and to Prof. K. SmMURA, this University, and Dr. T. KUaASAWA, Miyagi Prefecture Sericultural Experiment Station, for the donation of silkworms.

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effects of 3β-(β,β-dimethylaminoethoxy)-steroids on sterol metabolism and development of bombyx...

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