Sponge Secondary Metabolites: Biochemical and ...

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from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulat- ing the bacteria with which the sponge is symbiotically.
0022-1554/86/$3.30 The Journal of Histochemistry Copyright © 1986 by The

and Cytochemistry Histochemical Society.

Vol.

CORNELIUS Institutfur mora

MULLER,2

SOBEL,

Physio/ogische

i oceanografiju,

Received

for

BARBEL

Kotor

publication

BECHTOLD,

Universitdt,

Yugloslavia

February

and

and

6500

Institut

in revised

form

May

avarol, a potent cytostatic and hydroquinone, is present in large amounts only in the sponge Dysidea avra (2.7 g avarol/1 kg of fresh material). The present study was designed to determine the storage site of this compound within the organism. Light and transmission electron microscopic studies revealed that avarol is probably stored only in spherular cells. The compound is compartmented in intracellular cytoplasmic vesides in a paracrystalline form, and therefore can have no inhibitory effect on the sponge

are the simplest

a state

of

of an

of fusion

live

bacteria

(Muller

hypothesis

that cells

and

symbiosis

et al. , 1986).

the

symbiotic

sponge al.,

Dysidea

1985b),

agent dure,

which

Stniegler,

limited with

has its foundation

contains

avara was

fungi,

low

level

the

fact

that

bryozoae,

evidence between

and

supports sponge

a growth-promoting

principle we discovered

(cytostatic that the

amounts to be

in

This

in both

large

determined

by

algae,

sponge

coexisting

1979).

Experimental relationship

(e.g., lectin) and a growth-inhibiting (Muller et al. , 1981a). Recently,

A single

organisms

is additionally

in obligate

non-sponge

organisms.

of individual

(Weissenfels

individualization

sponges

multicellular

aggregate

of avarol

a highly

the and agent) marine

(Muller

active

KLJAJK,

West

Universitdt,

14,

accepted

1986;

cytostatic

May

metabolites;

Spherular

influence

Correspondence

iologische Germany.

Chemie,

to: Prof.

Dr.

Universit#{228}t,

W.E.G. Duesbergweg,

Muller,

Institut 6500

f#{252}r Phys-

Mainz,

West

biologiju

(AD).

(6A0663).

cells;

Sponges.

natural

product.

age of avarol in Dysidea avara. dence that avarol is synthesized been

shown

pounds

that

these

(MUller

et

in the

probable

present

study

temporary

ston-

can

contain

biologically

Thompson

et

al.,

cvialready

active

com-

1984).

Methods

collected

of the

marine

by dredging

sponge

in the

Dysidea

Bay of Kotor

avara

(Dictyocer-

(Yugoslavia)

in Sep-

1985.

Avarol.

This

compound

(MUller et al., of avarol, see

They

thoroughly

from

Rr electron ofsponge

2% specimens were

tetroxide,

hr. After

dehydration,

in 0.05

then

cut

into

in the small

in cacodylate 1%

in 0.05

cubes

M cacodylate

the specimens

were

as previously and chemical et al. (1985b).

preparations

1 cm thick

buffer,

fixative

buffer.

avara

phyiscal MUller

microscopic about

M cacodylate

remained

overnight

osmium

pieces

Dysidea

For details of the et al., (1974) and

fresh

in glutaraldehyde, The

was isolated 1985b). Minale

of Sections. sections,

sucrose. ature.

and

We provide some experimental in spherular cells. It has

1981b;

Specimens

were

tember,

cells

al.,

and

Animals. atida)

Therefore,

the site of synthesis

Preparation

2

1986

Germany

has a potent cytostatic effect on eukaryotic cells, the sponge protects itself against the inhibitory

of this

we determined

semi-thick Supported in part by a grant from the Deutsche Krebshilfe e.V. and by a grant from the Bundesministerium f#{252}r Forschung und Technologie (German-Yugoslavic cooperation program) under the coordination of the Internationales B#{252}no GKSS, Geesthacht.

24,

West

u

described properties

I

Mainz,

DORN

CS, AB); Zavodza

BD-S,

cells. Quantitative analysis utilizing high-pressure liquid chromatography revealed that avarol is present at a concentration of 3.2 tg/1O6 spherular cells. It appears that avarol is released from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulating the bacteria with which the sponge is symbiotically associated. Hisrochem Cyrochem 34:1687, 1986) KEY WORDS: Avarol; Antimitotic agent; Dys:dea avara; Secondary

(Muller et al., 1985a). Using the appropriate isolation proce2.7 g of avarol can be isolated from 1 kg of fresh material.

(WEGM)

AUGUST

(iVEGM, 6500

Materials et

and

Germany

Because avarol we must ask how

Introduction is composed

Mainz,

f#{252}r Zoologie,

The secondary metabolite antibacterial sesquiterpenoid

Sponges

1986

USA.

Biochemical of the Antimitotic

ZORAN

Duesbergweg,

(ZK);

3, 1986

1687-1690,

DIEHL-SEIFERT,

ANDREAS

Chemie,

12, pp.

Article

Sponge Secondary Metabolites: and Ultrastructural Localization Agent Avarol in Dysidea avara1 E. G.

No.

Pnntedrn

Original

WERNER

34.

Inc.

pH

for 3 days of about

embedded

7.4,

and

pre-fixed with

at room

1 mm

Post-fixation buffer,

were

and

0.2 washed

was carried pH

7.4,

in araldite.

M

temperout

at 4’C Thin

in

for

2

sec-

1687

1688

MULLER,

lions

DIEHL-SELFERT,

A)

(700

studied tim)

with were

stained

Isolation and

were

fixed

.. .#-

.

: .

S

centrifuged The

model

ODS cm)

Hypensil filled

the

the

H20

under

injected;

(5 tm;

with

methanol: the

and

(85:15,

peaks

molar

same

5000

was

the

the

organic

in a Gynkotec wavelength

steel

detec-

column

filled (0.3

Elution

was

performed

done

at 300

nm.

was

proportional

coefficient

g) were mm,

a pre-column

used.

Detection were

30

and

analyzed

a variable

In addition,

material

for

g; 20CC)

cm stainless

25

Shandon). v/v).

gradient

cells (0.5

stirring

was

with x

1 cm cubes

dissociation, density

Spherular x

obtained

a 0.3

of avarol

extinction

mechanical

After

equipped

65)

and (0.5

observations.

Ficoll

of Avarol.

material

citrate

1979).

acetate. mm;

DORN

sections

was cut into

by discontinuous

(10

lead

microscopic

After

Konigsmann,

of ethyl

ton (Beckman 0.5

#{149}c

was

ml

chromatograph

planes

S

0.5

was collected.

with

qq:;t.

and

300 CS liquid with



obtained

and Identification with

suspension

.

were

for light

water.

and Semi-thick

The fresh sponge

formaldehyde-sea

cells

Extraction

phase

blue

Cells.

KLJAJK,

acetate

microscope.

methylene

(Bretting

extracted

-‘a

with

in 3.5%

the spherulan

in uranyl

EM 9A electron

ofSpherular

centnifugation

.

double-stained

a Zeiss

BECHTOLD,

SOBEL,

to the

at 300 nm

x

The

concentrations

was determined

to be

3820.

Results , ..

----

-

----:--

0

‘a.

...

-

i.

.

#{182}, .

..,

Earlier

studies

contain

either

(Muller .

had

already

et al. , 1981b).

.Th

Figures 1 and 2. Spherular cells of Dysidea avara. Figure 1 . Semi-thick section through the mesohyl. Arrows markthe spherular cells(light micrograph)Original magnification x 650. Figure 2. Isolated, whole spherular cells (light micrograph) Original magnification x 750. Bars = 20 tm.

cells

of

Dysidea

revealed

antibiotics

avara

that

spherular

(Thompson Therefore,

also

et

we wished

contain

the

al. ,

cells 1983)

to determine

secondary

in sponges or

lectins whether

metabolite

avarol.

Figures 3-6. Transmission electron micrographs of spherular cells. Figure 3. cell with vacuoles, containing lamellar (f) or granular (g) inclusions. Original magnification x 5400. Figure 4. Inclusions are packed as paracrystalline material (p); n, nucleus. Original magnification x 8500. Figure 5. Higher magnification of the paracrystalline inclusion content (p). Original magnification x 17,000. Figure 6. Release of the paracrystalline material (arrow); n, nucleus. Original magnification x 5000. Bars 2 tm.

=

OF AVAROL

LOCALIZATION

Light

Microscopic

Semi-thick

cells

ranges and

very

9-15

appearance

1.5-4.0

in the

After

almost

the

Their

step, They

size

of

1) that

diameter

dissociation

population.

2);

(Figure

mesohyl.

a mechanical centnifugation

pure

(Figure

revealed

the

proce-

these

have

cells

can

a mulberry-

vesicles

ranges

from

p.tm.

Transmission

Electron

After

sectioning,

cells

became

fibnillan .tm

of

cases,

the

documented

avarol

for the

tract

from

only

one

This

component

ofthe

that

vary

cells

(Figures

assume

contain

internal

4 and

5).

inclusions these

or

between

In

rare

could

internal

be

“grains” Figure

existance

spherulan major

of avarol

cells

was

analyzed

with

3.2

it cannot

contain

7). and

at a detection

co-eluted that

in spherulan

(Figure

component

However, also

We

vesicles

of single

chromatography

revealed

cells.

the

spherulan

granular

paracnystals.

by liquid

analysis

30%

contents

6).

in the either

of Avarol

proof

tamed

of

structure

of the

Identification One

About

vesicles

containing

diameters

3-6).

(Figure

represent

3),

a panacrystalline

release

Studies

cytoplasmic

(Figure The

(Figures

“grains”

Microscopic

intracellular invisible

inclusions.

0.3-4

cells

sponge

abundant

gradient

as an

H

the

tm.

a subsequent

be obtained like

through

are

between

dune

Observation

sections

spherular

1689

.tg

The

ethyl

determined

authentic

at present

from

Tethya

ob-

tions

ofthe

cx-

is present

to contain of

avarol.

is present

was

acetate

wavelength

of avarol

be excluded

cells

in

that

300

8. Chemical

nm.

(MUller

is usually

sponge

In

the

In

1955,

cytostatic

Bergman

and

agents;

Bunk

discovered

that

1--D-arabinofuranosylthymine

sponges was

that

contain

cells

that

live

in

(Bergquist,

1978).

quantitative

analysis

DNA

100

5 x

lated

to be localized

C

is apparently this

C’)

na

presented

This

in the

means,

that

in the

sponge,

because

As found paracrystals

0

2

4

6

8 10

‘12 14 16 18

20 22 24 26

t (mm) Figure 7. Identification of avarol pressure liquid chromatography.

in spherular Standards:

bly

represent yet

lan

space.

species cells of Dysidea avara by high1, authentic avarol; 2, avarone.

know

cells

avarol, the

are

However,

(Alcaligenes

sponge (in preparation), regulating the bacterial

is present

fact

sponge that

by bacteal-

unable

strain

to

that

we

results). (Donadey,

that

very

manner. in the only

extracellulan

it is possible that flora. This suggestion

1982),

which

ofavarol

in the

the

calcu-

1 kg Dysidea

were

in a menocnine

the

From was

be produced

we

avara,

ifany,

of

unlikely

a bacterial

sponges

of Dysidea

considering sp.)

to

(unpublished

are 10i2

Conse-

in the

It is very

known

from

other

role,

in

present

Moreover,

broth

released

functional

10%.

8) is synthesized

are

cells.

avara

present

avanol cells.

terpenes

in some

in spherular

not

cells

all the

x

percentage

be

1.6 g of avarol

by eukaryotic

already

The to

cells

0.16

in 100 g oftissue.

(Figure

detect any avarol in a culture have isolated from Dysidea

cell;

in Results,

compound

exclusively

mg).

semi-

lO

cells exist spherular

as cells

content,

pen

80

The

microgranular

5 x

estimated

in spherulan

sesquiterpenoid

most

tissue;

almost

sequestered

classified

DNA

content

was

spherular

avarol.

be

approximately

(DNA g of

as

with

identified

to be

can

of

evidence bacteria

were

interpreted

basis

tissue

of Streptomyces

cells

appear

that

of fresh compound

by the

“grains”

the

100

the

data

E 0 0

per in

not

particular,

they

revealed

lOb

experimental

cases

deter-

0.3% active

experimental

and

were

agent

We

al. , 1969).

some

internal

g of sponge

cells

quently,

these

rare

amounts.

broths

cells

investiga-

a cytostatic

comprises

et

provide

In

On

content

avara.

we

these

isolated

spherular

avarol

fermentation

symbiosis.

in

high

(Bernard

paracrystals;

cells;

in our

that

of a biologically

by sponge

contain

in

that

in

study,

spherulan

g;

1985b)

only

contain

present

in extremely

is produced

they

surprised

to discover

antibiotics

present

avarol

we were

a concentration

obtained

which

Discussion

et al.,

producing

that

However,

organism

Such

strains

of avarol.

Dysideaavara

in this

mined

Quantitative

other

crypta. sponge

materials.

106 spherular

avarol.

structure

the

probaWe

do

extracellu-

one space

bacterial of this

avarol is involved in is supported by our

1690

MULLER,

recent

finding

that

avarol

antifungal activity In preliminary cytostatic preparation). tected basis

arrest

in some

way against

Moreover,

in

by

these a form

penimental

data cells

the most

Literature

a

of avarol. avarol special

avarol

functional the

is stored

mechanism

is binding

as

No

by which

avarol. ofthe

the

vacuoles.

inertness.

extracellular

or soluble

On

is func-

cxthe

At present,

compound

to cx-

macromolecules.

Cited

PR (1978):

Sponges.

H, Konigsmann

cells in the sponge

Axinella

Contribution of sponges. London,

C, Manal

M,Jacquillat

K (1979):

to the study Chem

Investigations

polypoides

ofmanine 20:1501

J Org Hutchinson

University

R (1969): on the

(Schmidt).

Donadey C (1982): Les cellules pigmentaires l’#{233}ponge Cacospongia scalaris (Demosponge 4:67

and causes

effect within

about

explanation

BernardJ, Paul R, Boiron Berlin, Springer-Verlag Bretting

avarol

intracellular

organelles

against

Bergman W, Bunk DC (1955): ucts; XXXIX. The nucleosides Bergquist

that

guarantees

are available

structures

that

cytostatic

cytoplasmic

are protected

plausible

tracellular

the

sequestration

that

antibacterial

cells of Dysidea avara in vitro (in the sponge cells in vivo must be pro-

it is likely

inactivated

paracrystals, sponge

data,

considerable

et al., 1985). we determined

of dissociated Consequently,

of available

tionally

displays

(Seibert studies,

prod-

Rubidomycin.

lectin-producing

Cell Tissue Res 201:487

et les cellules Dictyocenatide).

a inclusions

de Vie Marine

KLJAJI, DORN

BECHTOLD,

Minale L, Ricco R, Sodano G (1974): Avarol, a novel sesquiterpenoid hydroquinone with a rearranged drimane skeleton from the sponge Dysidea avara. Tetrahedron Lett 38:3401 Muller WEG, Maidhof A, Eich E, Schn#{246}der HC (1986): Potent kemic and antimicrobial activity of the novel antimitotic agents and avarol. In Proceedings of the First International Congress Adjuvant Chemotherapy. Paris, Nov 7-9, 1985, in press.

antileuavanone on Neo-

MUller WEG, Maidhof A, Zahn RK, Schn#{246}der HC, Gasif MJ, Heidemann D, Bernd A, Kurelec B, Eich E, Seibert G (1985a): Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo. Cancer Res 45:4822 Muller WEG, Zahn RK, Gasif Diehl-Seifert B, Eich E (1985b): from the marine sponge Dysidea

MJ, Dogovif N, Maidhof A, Becker C, Avarol, a cytostatically active compound avana. Comp Biochem Physiol 80C:47

Muller (1981a): sponges.

WEG, Zahn RK, Kurelec Lectin, a possible basis J Bacteniol 145:548

MUllen (1981b): tification

WEG, Zahn RK, Muller I, Kunelec B, Uhlenbruck G, Cell aggregation of the marine sponge Geodia cydonium. of lectin-producing cells. EurJ Cell Biol 24:28

Seibent Library

DIEHI,-SEffERT, SOBEL,

(1985):

Bakt

G, Raether Antibacterial Hyg 260A:379

B, Lucu for

W, Dogovi N, and antifungal

C, MUller

symbiosis

between

I, Uhlenbruck bacteria

G and

Vaith

P

Iden-

Gasif MJ, Zahn RK, Muller WEG activity of avarone and avanol. Zbl

ThompsonJE, Barrow KD, Faulkner DJ (1984): Localization oftwo brominated metabolites, aerothionin and homoaerothionin, in sphenulous cells of a marine demosponge, Aplysina fistularis (Verongia thiona). Acta Zool 27:410

Weissenfels schwammes problem.

N,

Stniegler

Ephydatia Zoomorphol

B

(1979):

fluviatilis 92:49

Bau

und

L. (Ponifera).

Funktion VI.

Das

des SUsswasserIndividualit#{228}ts-