Enzymatic transformation of PSP toxins in the littleneck clam (Protothacastaminea )

Vol. 114, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

July 29, 1983

Pages

ENZYMhTIC TRA.KSFORMATION OF PSP TOXINS THE LITTLENECK CLAM (Protothaca staminea) John J. Sullivan, Institute

Received

June

13,

Wayne T. Iwaoka

465-472

IN

and John Liston

for Food Science and Technology University of Washington Seattle, Washington 98195

1983

SUMMARY: Stemming from investigations into the relationship between toxins produced by Gonyaulax sp. and accumulated in shellfish, we wish to report enzymatic transformations of the PSP toxins to decarbamoyl derivatives in No toxin transformations were the littleneck clam (Protothaca staminea) . observed in either mussels (Mytilus edulis) or in butter clams (Saxidomus giganteus). In addition, littleneck clam samples from the natural environment contained predominantly the decarbamoyl derivatives, while other shellfish species collected from the same vicinity contained the previously reported PSP toxins. Paralytic temperate

shellfish

coastal

neurotoxins

it

areas

it

(GTX I-IV) parts

that

that

several

and neosaxitoxin

of the world

PSP toxins

(see strains

as the N-sulfocarbamoyl these that

toxins the

have

number

butions

have noted

between

dinoflagellates

of shellfish closely be the

recently

of related

related reason

differences

and shellfish in

the

metabolic in

shellfish is

relative

interconversions toxin

1)

(8).

It

profiles.

(5-7), is

and

toxin

involving distri-

of the various different

the toxins

species

are

of the Recently,

toxins

all

toxins

All

Copyrighr 0 1983 rights of reproduction

may

transfor-

0006-291X/83

465

that

apparent

complex,

addressing

Since

reported

predominantly

10) and between (3).

from many

has been

extremely

levels

was involved,

shellfish

in figure

studies

(9,

the same area

dissimilarity

in

Several in the

occur

B2, Cl-C4

in shellfish

toxins.

structurally, for

(Bl,

been reported

of toxicity

collected

sp.

it

molluscs.

the gonyautoxins

in toxic

Recently

of potent

(STX),

toxins,

present

Gonyaulax

to many of the

in bivalve

saxitoxin

related

1).

derivatives

development

a large

figure of

sp.)

one toxin,

are

endemic

the accumulation

(Gonyaulax

closely

(NEO),

(l-4)

in several

only

a problem

involves

origin

was thought

is now known

(PSP),

of the world,

of dinoflagellate

Originally but

poisoning

$1.50

by Academic Press, Inc. in any form reserved.

BIOCHEMICAL

Vol. 114, No. 2, 1983

AND BIOPHYSICAL

P.S.P.

RESEARCH COMMUNICATIONS

Toxins Carbamate Toxins

R3 Figure

mations

(R4-SO;)

H

STX

Bl

ON

tl

N

NE0

ON

I

OSOi

GTX

I

C3

N

H

OSOj

GTX

II

Cl

II

oso;

H

GTX

III

c2

OH

OS&

H

GTX

IV

c4

02

the conversion

of the gonyautoxins

(11).

reported We wish

of the PSP toxins

and on the distribution

species

in Puget

Sound,

in one species to report

in littleneck

genates

MATERIALS

(R4-H)

R2 II

with paralytic shellfish poisoning NEO-neosaxitoxin, STX-saxitoxin). have not been reported but are postulated forms of GTX-I and GTX-IV respectively.

have been

maqellanicus)

R3 Rl N

Toxins associated (GTX-gonyautoxin, Toxins C3 and C4 N-sulfocarbamoyl

involving

saxitoxin

sion

1.

N-Sulfocarbamoyl Toxins

of scallop

on the occurrence clam

of

and neosaxitoxin

(Protothaca

the toxin

metabolites

to

(Placopecten of metabolic staminea)

conver-

tissue

in various

homo-

bivalve

Washington.

AND METHODS

Toxic and nontoxic littleneck clams (Protothaca staminea), mussels (Mytilus edulis), and butter clams (Saxidomus giganteus) were collected from beaches on Puget Sound, Washington. All samples were either processed immediately after collection or held live for a short time in aquaria (12OC) until utilized. Investigations into the metabolic transformations of the PSP toxins were conducted by incubating purified toxins in tissue homogenates (Xg tissue + 6X ml H20, pH 6.2) at 30°C for periods of time ranging up to 24 hr. At the end of the incubation period, enzymatic activity was stopped by adding 4 volumes 95% methanol (pH 4.0). The toxin content before and after incubation was determined by high pressure liquid chromatography (HPLC) using both amino and cyano bonded phase columns (12). Decarbamoyl derivatives of the PSP toxins were prepared chemically by strong acid hydrolysis (7.5 N HCl) (13) and carbamoylation reactions were carried out according to the procedures described by Koehn et al. (14). Toxicity testing of the purified toxins and tissue homogenates was performed by the mouse bioassay (15) using 19-22 g Swiss-Webster mice. The occurrence of the various PSP toxins in the natural environment was investigated by analyzing dinoflagellates and naturally contaminated shellfish from Puget Sound, Washington. Shellfish samples were extracted with 2 volumes .05N HCl, an aliquot of the supernate diluted with an equal volume metanol, and analyzed by HPLC as previously described. A similar procedure was used for dinoflagellate cells (collected in a 28 urn net) except acetic acid was substituted for HCl.

466

Vol. 114, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION Incubation

of the N-sulfocarbamoyl

clam homogenates

effected

reaction

were

reported

to date,

column

than

compounds

showing

were

formed

muscle,

the same products carbamate

neck

STX-M,

GTX-IIM

in

4 hours)

(STX-M,

PSP toxins

(STX,

is

somehow

transformation vation

is

low pH effectively mussel

block

homogenates,

N-sulfocarbamoyl the

in

the

that

carbamate

toxins.

rates

(figure

reaction.

organic

solvents

even after

recovered

in

group

the on the

Evidence

that

heat

this

inacti-

(methanol),

In the case of the

were

of little-

than

the sulfo

that

in that

by incubation 2, A) with

the observations

clam

was found

was much slower

occurred

and C2

of formation

it

formed

enzymatic

toxins

transformations

have

toxicities The reported

of toxin

needed

metabolite

described intermediate

1050 and 2300 MU/pmole

for

Cl,

the new

of the littleneck

that

reaction.

15, 150,

toxin

were

that

butter

or

clam and

24 hr and the

intact

at the end of

period.

compounds

STX-M were

from Bl,

In addition,

adding

of the

on the amino

was found

suggesting

no transformation

The enzymatic

MH/pmole

It

GTX-III)

with

(boiling),

and carbamate

incubation

the

rests

time

decreasing

the reaction

involved

of the homogenates

with

GTX-IIIM)

toxins,

enzymatic

2).

littleneck

of the PSP toxins

in homogenates

GTX-II,

although

case of the N-sulfocarbamoyl

figure

C2) in

The products

retention

homogenates.

GTX-IIM,

all

and GTX-IIIM

rapidly

and siphon

clam homogenates,

carbamate

(see

Cl,

toxins.

from

longer

most

(ca 90% completion

the mantle,

the

toxins

(Bl,

of the

distinct

a somewhat

carbamate

(designated

respectively) viscera

transformation

chromatographically

the

toxins

to kill

(GTX-IIM)

content

of Cl,

Bl,

by correlating

are

the

reaction

same

as for

the 467

of

or MU is

toxicities

mixture that

carbamate

and

and STX are the

ca

amount

of the

are approximately

(The

by HPLC, and assuming the

GTX-II

The toxicities

(STX-M)

respectively.

in formation

the N-sulfocarbamoyl

(One mouse unit

and the Bl metabolite

as measured

the metabolites

toxicities (6,7).

result

between

a 20g mouse in 15 min.)

and 900 ? 120 MU/umole determined

above

Cl

380 + 50

of GTX-IIM toxicity

with

the fluorescence toxins.)

This

and the

yields has

Vol.

114,

No.

2, 1983

A

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

hr

24 hr

a

0 hr

!

dc STX

c ti :,

min

1’5

LL c, min lb

d

min

lb

Carbamoylation

Minutes Figure

2.

A: Transformation of STX (a) to STX-M (b) in homogenate of iittlenack clam visceral mass (30°C, pH 6.2, STX added at the level of 15 MM). Decarbamoyl STX (dcSTX) is shown below for comparison. Incubation of Bl forms the same product (STX-M) and reaction is essentially complete after 4 hr. HPLC conditions p Bondapak amino column (4 mm x 30 cm) with 62% MeOH, .03M ammonium phosphate mobile phase, flow 1.2 ml/min. B: Chromatograms of ETX-IIIM (f) produced

GTX-II from

(c), GTX-III a mixture of

468

(d), GTX-IIM (e) Cl and C2 by either

and acid

Vol. 114, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Toxin Metabolites

Bl: Cl:

c2:

Proposed Enzyme Pathway

.,

GTX Ml: GTX IIIM:

X=aOSO; X=oOSO,

STX: X=‘H GTX It: X=aOSO; GTX Ill: X=fiOSoi Figure

3.

important

implications

shellfish

possessing

those

without

the

thesis

includes:

removes Fig.

from a food these

the enzymes

The observed yielding

The metabolic transformations occurring clam involve hydrolysis of the carbamate the N-sulfocarbamoyl or carbamate toxins GTX-gonyautoxin).

for

(a) group

acid

from

toxin

hydrolysis

(see

hydrolysis

different

3).

(.ZN

HCl,

Support 100°C)

the carbamate

clam enzymes - Lichrosorb .04M ammonium

C: Mixture of the carbamate toxins (GTX-II/III and STX) and the metabolites (GTX-II/IIIM and STX-M) before and after carbamoylation, illustrating the conversion of GTX-II/IIIM and STX-M to GTX-II/III and STX respectively. This procedure was used to confirm that the metabolites are the decarbamoyl toxin derivatives. HPLC conditions -.as in B above, except mobile phase is 60% MeOH, 0.32M ammonium phosphate, flow 0.8 ml/min.

469

suggests toxicity

that from

content.

2 Continued.

hydrolysis (.2N HCl, lOO"C, 5 min) or by littleneck (incubation conditions as in A). HPLC conditions amino column (5 pm, 4 mm x 25 cm) with 50% MeOH, phosphate mobile phase, flow 1.1 ml/min.

it

of the

figure

Cl and Bl yielding

the littleneck group from either (STX-saxitoxin,

since

a vastly

total

involve

derivatives mild

standpoint

may have

any given

transformations

decarbamoyl

the sulfo

enzymes

safety

in

carbamate for [which toxins

ester

this

hypo-

effectively and a

BIOCHEMICAL

Vol. 114, No. 2, 1983 corresponding either

shift

in HPLC retention

HPLC retention

of a sulfo

group

decarbamoyl

attached

to hydrolyze

the

(X on figure

3) intact),

STX-M,

(c)

same for

toxicity

on both

times

STX and STX-M results

by their

(see

in the

reaction

studied

and STX as substrates,

it

likely

that

The occurrence investigated fish

Puget

of the various

Sound,

was found

to be mostly

enzymatic

conversions

in mussels gesting

closely

that

consistent Puget

Sound

and mussels possibly Puget toxins

usually

are being

is

Bl,

observed

columns

are

the

carbamoylation

Figure

Cl,

for

and STX respectively

littleneck

the enzymes

3 illustrates

clams.

While

C2, GTX-II,

involved

vitro

usually

GTX-III,

would

be active

in vitro the

occur

toxin

laboratory

profile

metabolically

are

four

of C2 and GTX-III mainly

STX,

clams

that

The toxin

in these

The toxin

toxins

of Bl,

the profile

plus

indicates

sugshellfish,

profile

in

C2, GTX-III

and STX,

Cl and GTX-II

respectively).

which

converted

vivo.

shell-

littleneck

suggesting

occurring

predominantly

these

in

Butter that

either

to STX (as has been

shown

(due

clams

in

the

other

in

(1) The hydrolysis of the sulfate (X in figure 3) from GTX-II, yielding ll-hydroxy saxitoxin, has been shown to have no effect on toxicity (ca 1000 MLJ/pmole), while the decarbamoyl 11-hydroxy saxitoxin has toxicity of 320 MU/pmole, which is very close to that found for GTX-IIM (G. Boyer, Ph.D. dissertation, U. of Wisconsin, 1980). 470

was

of the dinoflagellates,

observations.

consists contain

in

environment

contaminated

composition

and STX-N,

transformations

contain

in the natural

and naturally

GTX-IIIM

observed

to epimerization Sound

using

The toxin

GTX-IIM,

dinoflagellates will

2C).

method

group

and (d)

of GTX-II

of the

of a suitable

to that

2A),

absence

toxicities

and cyano

in the

the

saxitoxin

similar

figure

PSP toxins

Washington.

resembles

in

is

dinoflagellates

no metabolic with

the

in

PSP toxins.

by analyzing

from

sulfate

occurring been

reported

leaving

see figure

has only

on the other

lack

formation

transformation

is

the

(d ue to the

the amino

HPLC behavior;

enzymatic

(b) while

which

no change

indicating

of decarbamoyl

the retention

(as determined

this

while

(13),

and STX-M

the proposed

moiety

1200 f 300 MU/pmole

decarbamoyl

of GTX-IIM

carbamate,

are unknown (I)

the

causes

of the metabolites,

at the

carbamate

RESEARCH COMMUNICATIONS

and toxicity]

or toxicity

gonyautoxins

reportedly

AND BIOPHYSICAL

Vol. 114, No. 2, 1983 scallops)

(ll),

since

BIOCHEMICAL

or there

the dinoflagellates toxins.

a similar

HPLC retention

column)

available did

determine

retention

We have

toxins.

It

(11)

that

Yoshioka important

role

develops

that

littleneck is

apprent

derivatives

different

from

since

the other

in the

in the

toxicity

toxicity

of the

decarbamoyl

in naturally

of the

important development

study

of these

samples

were

possible

to determine

be necessary but

it

is

to

evident

that

conversions

and

of the PSP toxins enzymatic

and those

profile

decarbamoylation

of Shimizu

of the PSP toxins

indicate

a very

toxicity

that

that

and N-sulfocarbamoyl a very

important

and

may play

and overall also

enzymes

of toxicity

It

toxin part

in addition forms,

of the

littleneck

the

toxin

clams

of the end products is

evident

toxins

toxicity.

that

are

the

hydrolysis

by their

pro-

shellfish

471

toxins

and a to a

the presence and the

processes

and may partially

quite

transformations

of the carbamoyl

of the biochemical

have

conversion

Determining

contaminated

in shellfish

the

of the N-sulfocarbamoyl

carbamate

catalyzing

to an understanding

in

toxicities

PSP toxins.

of intermediate

bution

in

metabolic

involves

occurring

the

common end product toxins

detected

species.

in an increase

decrease

this

investigations

transformations

implications

on the amino were

transformations

toxin

carbamate

with

the siphon.

this

may represent

in some shellfish

result

the

clam,

both

conversions

These reported

important

will

in

from

metabolic

The metabolic

investigations

clam and that

in shellfish.

decarbamoyl

was not

metabolic

in determining

to the previously

it

to

a toxin

late

quantities

involving

of some toxins

clam,

transformations

peak,

clam

of STX in relation

slightly

in the butter

complex,

demonstrated

in the

of the

occurring

are very

(eluting

no toxin

Further

quantity

in the butter

and insufficient

STX-M.

RESEARCH COMMUNICATIONS

of STX in the butter

a small

the unknown

the processes

selective

file

Since

clam homogenates

the mechanisms

only

to STX-M

observed.

represent

retention

to saxitoxin

time

to characterize

it

occur

In addition

was often

the butter

selective

produce

the other

if

is

AND BIOPHYSICAL

species

moiety involved

explain

of the distri-

are in the

the vast

Vol. 114, No. 2, 1983

differences dinoflagellate

in

toxicity

BIOCHEMICAL

often

AND BIOPHYSICAL

observed

between

RESEARCH COMMUNICATIONS

shellfish

species

during

blooms.

ACKNOWLEDGMENTS This research was supported in part by a fellowship Research Fund. We wish to thank Sherwood Hall for purified dinoflagellate toxins.

from the Egtvedt Food the generous supply of

REFERENCES 1. Hsu, c. P., A. Marchand and Y. Shimizu (1979) J. Fish. Res. Bd. Canada 36, 32-36. 2. Noguchi, T., Y. Ueda, K. Hashimoto and H. Seto (1981) Bull. Jap. Sot. Sci. Fish. 47, 1227-1231. 3. Shimizu, Y., W, E. Fallon, J. C. Wekell, D. Gerber and E. J. Gauglitz (1978) J. Agric. Food Chem. a, 878-881. 4. Shimizu, Y. (1979) in Toxic Dinoflagellate Blooms, pp. 321-326, D. L. Taylor and H. H. Seliger, Eds. (Elsevier-North Holland). 5. Hall, S., P. B. Reichardt and R. A. Nev6 (1980) Biochem. Biophys. Res. Comm. 97, 649-653. Fix Wichmann, C., W. P. Niemczura, H. K. Schnoes, S. Hall, P. B. 6. Reichardt and S. D. Darling (1981) J. Am. Chem. Sot. 103, 6977-6978. 7. Koehn, F. E., S. Hall, C. Fix Wichmann, H. K. Schnoes and P. B. Reichardt (1982) Tet. Letters 23, 2247-2248. 8. Onoue, Y., T. Noguchi, J. Maruyama, K. Hashimoto and T. Ikeda (1981) Bull. Jap. Sot. Sci. Fish. 67, 1643. 9. Oshima, Y., W. E. Fallon, Y. Shimizu, T. Noguchi and Y. Hashimoto (1976) Bull. Jap. Sot. Sci. Fish. 42, 851-856. 10. Onoue, Y., T. Noguchi, J. Maruyama, Y. Ueda, K. Hashimoto and T. Ikeda (1981) Bull. Jap. Sot. Sci. Fish. 47, 1347-1350. 11. Shimizu, Y. and M. Yoshioka (1981) Science 212, 547-549. 12. Sullivan, J. J. and W. T. Iwaoka (1983) J. Assoc. Off. Anal. Chem. 66, 297-303. 13. Ghazarossian, V. E., E. J. Schantz, H. K. Schnoes and F. M. Strong (1976) Biochem. Biophys. Res. Comm. 68, 776-780. 14. Koehn, F. E., V. E. Ghazarossian, E. J. Schantz, H. K. Schnoes and F. M. Strong (1981) Bioorg. Chem. lo, 412-428. 15. Methods of Analysis, 13th Ed., Assoc. Offic. Anal. Chem., Washington, D.c.935).

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