Cadmium-binding proteins in the blue crab, callinectes sapidus: laboratory-field comparison

Marine Environmental Research 14 (1984) 139-151

Cadmium-Binding Proteins in the Blue Crab, Callinec tes sap idus: Laboratory-Field Comparison

David W. Engel

National Marine Fisheries Service, NOAA, Southeast Fisheries Center, Beaufort Laboratory, Beaufort, North Carolina 28516, USA

&

Marius Brouwer

Marine Biomedical Center, Duke University Marine Laboratory, Beaufort, North Carolina 28516, USA

ABSTRACT

The blue crab, Callinectes sapidus, is distributed along the east coast of the United States from Cape Cod, Mass., through the Gulf of Mexico, including both relatively unpolluted coastal areas and estuaries contaminated with trace metals. Cadmium is of particular concern because it is concentrated in the digestive glands of blue crabs and can be passed on to consumer organisms. Tissue concentrations and partitioning of trace metalsJrom crabs exposed in the laboratory to lOppb dissolved cadmium for 40 days were compared with blue crabs collected from two locations on the Hudson River, NY, Foundry Cove and Haverstraw Bay, both of which have elevated trace metal levels relative to estuarine areas near BeauJbrt, NC. Crab digestive glands, gills and muscle were removed and analyzed for total cadmium, copper, zinc and nickel concentrations using acid digestion and atomic absorption spectrophotometry, and metal-binding (metallothionein-like) proteins were determined by gel filtration chromatography. In crabs exposed to cadmium in the laboratory, the cytosolic partitioning was similar to previous investigations at our laboratory where higher levels of cadmium ( lOOppb) and shorter exposure times (14 days) were used. The similarity in cadmium partitioning from these two separate experiments indicates dose independence. In crabs from polluted environments the digestive glands contained the

139 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain.

140 David W. Engel, Marius Brouwer

highest concentrations of trace metals. Chromatograms of the cytosol from the digestive glands and gills from both field and laboratory- exposed crabs showed similar distributions of cadmium, copper and zinc. The gills of both field and laboratory-exposed crabs had metal-binding proteins that contained mostly cadmium, and the digestive glands had metallothionein-like proteins that contained cadmium, copper and zinc. Estimated molecular weights Jor these proteins were similar to the metallothioneins jound in other crustaceans and mammals.

INTRODUCTION

The blue crab, Callinectes sapidus, an ecologically and commercially important estuarine crustacean, is distributed along the east coast of the United States from Cape Cod, Mass., through Florida and the Gulf of Mexico. Owing to its wide geographical distribution and physiological adaptability, it has been studied intensively. Its estuarine dependence gives it a high probability of being exposed to elevated levels of pollutants.

One of the more contaminated systems in the northeastern United States is the Hudson River-Raritan Bay estuarine system in New York where trace metal levels are high (Breteler, in press) compared to the Newport River estuary at Beaufort, NC (Evans, 1977). Accordingly, two sampling locations were selected on the Hudson River: Foundry Cove and Haverstraw Bay. Foundry Cove sediments have high concentrations of cadmium and nickel attributable to an abandoned aircraft battery plant (Kneip & Hazen, 1979). Haverstraw Bay sediments are high in cadmium from non-point sources, and in copper, presumably from the Indian Point electric power generating plant (personal communication, J. O'Connor, New York University Institute of Environmental Medicine, Tuxedo Park, NY).

Recently we have demonstrated cadmium/zinc and cadmium-binding proteins in the digestive glands and gills of blue crabs exposed to cadmium in the laboratory (Brouwer et al., this volume, pp. 71-88). These proteins, whose presence has been demonstrated repeatedly, have a molecular weight and amino acid composition similar to mammalian and crustacean metallothioneins and metallothionein-like proteins* that have

* The use of the term'metallothionein-like protein' is predicated on the fact that unless the molecule has been purified and characterized and is similar to mammalian metallothionein in amino acid composition, it is inappropriate to call such a protein metallothionein.

Cadmium-binding proteins in the blue crab 141

been studied (Overnell & Trewhella, 1979; Olafson et al., 1979; Roesijadi, 1981 ; Ridlington et al., 1981). Wiedow et al. (1982) also demonstrated the presence of cadmium-binding proteins in blue crabs collected from the Hudson River.

Since blue crabs from the lower Hudson River were known to have elevated concentrations of cadmium (Wiedow et al., 1982) and since we had already partially characterized blue crab metallothionein-like proteins from laboratory-exposed crabs, experiments were designed to compare metal-binding (metallothionein-like) proteins from laboratory and field-exposed blue crabs. The tissues chosen for analysis were the digestive glands and gills, which had been shown in laboratory experiments to contain the majority of the total body burden of cadmium in the blue crab (Engel, 1983).

MATERIALS AND METHODS

Blue crabs used in this investigation were exposed either to cadmium in the laboratory or to cadmium in contaminated environments. Crabs exposed in the laboratory were collected from the estuary in the vicinity of Beaufort, NC. Those exposed in the environment were collected from the Hudson River at Foundry Cove and Haverstraw Bay.

Laboratory exposures were conducted in a flowing seawater exposure system (Engel & Fowler, 1977). The cadmium levels were maintained at 10 ppb for 40 days. During the exposure period, crabs were fed clams and chopped squid every other day. Salinity ranged from 30 to 33%0, and temperature was 20 °C.

All crab tissues were sampled in a similar manner. After a crab was killed, the digestive gland, gill and muscle tissue were quickly dissected out. Half of each individual tissue sample was frozen at - 20 °C~and saved for determination of metal-binding proteins. The remainder was placed in preweighed beakers and analyzed for concentrations of cadmium, copper and zinc.

Soluble metal-binding proteins from crab tissues were prepared from homogenates and analyzed by gel chromatography according to the modified procedure of Ridlington & Fowler (1979). Digestive glands and gills were homogenized at high speed in a Brinkman Polytron*

* The use of trade names does not indicate product endorsement by the National Marine Fisheries Service, NOAA.


homogenizer in the presence of 1.5 volumes of 10 mM phosphate buffer plus 0.1 M NaC1 at pH 8 and 5 x 10-4M phenylmethyl sulfonylfluoride (PMSF) at 4 °C, and then centrifuged at 30 000 x g for 30 min at 4 °C. The supernatant was heated at 60 °C for 10 min, cooled in ice for 1 h, and then centrifuged at 30000 × g for 30min before being applied to the chromatographic column.

In chromatographic separations, Fractogel HW-55F was used as the gel filtration medium. The eluant was 10 mM phosphate buffer plus 0-1 M NaC1 at pH8. Fractions were monitored spectrophotometrically at wavelengths of 280 and 254nm and their trace metal contents were measured by atomic absorption spectrophotometry and flame aspiration.

Proteins of known molecular weight were used to standardize the chromatographic columns. The calibrating proteins were catalase 232 000 Mr,* aldolase 158 000 M r, bovine serum albumin 67 000 M r, ovalbumin 43000 Mr, chymotrypsinogen A 250000Mr, myoglobin 17 000 Mr, ribonuclease A 13 700 M r and cytochrome c 13 000 M r. Flow rates for all separations averaged 30 ml/h and the column dimensions were 3.2 × 63cm.

Tissue samples were analyzed for trace metals by using standard atomic absorption spectrophotometric techniques. Samples were dried at 90 °C, wet-ashed in concentrated HNO3, and then diluted and analyzed by flame aspiration atomic absorption spectrophotometry.

The National Bureau of Standards Oyster Reference Material No. 1566 was used to calibrate the zinc, cadmium and copper measurements. The certified concentrations in the Standard Reference Material were for zinc 852 + 14, for cadmium 3.5 + 0.4, and for copper 63 _+ 3"5 ~g/g (_+ refers to the 95 % confidence interval around the mean value). Mean concentrations for 10 replicate aliquots of the Standard measured in our laboratory were for zinc 848__ 63, for cadmium 3.4 _ 0.4, and for copper 60.2 _+ 3.2 #g/g. Our mean values, therefore, are in agreement with the specified elemental concentrations.

RESULTS AND DISCUSSION

The elution profiles from tissues of blue crabs exposed to cadmium in the laboratory for 40 days showed distinct cadmium-copper -z inc and cadmium-binding proteins in the digestive gland and gills (Figs 1 and 2).

* M r means relative molecular mass.

Labo

rato

ry E

xpos

ed

2'0 fl.0

__

280n

m

~ A

0.5t

0

6~0

7L0

80

9LO

160

FRAC

TIO

N NU

MBE

R E

lutio

n pr

ofile

of t

he cy

toso

lic f

ract

ion

from

the

gills

of

er

2.0

0 1.0

0 ~

o.5

~ o 30

40

50

Fig

. 1.

bl

ue c

rabs

exp

osed

in th

e la

bora

tory

to 1

0 ppb

cad

miu

m in

the

wat

er f

or 4

0 da

ys.

Eac

h el

utio

n pr

ofile

is

repr

esen

tativ

e of

the

chro

mat

ogra

ms

prod

uced

fr

om

5 in

divi

dual

bl

ue

crab

s.

Sepa

ratio

ns

mad

e on

a

Frac

toge

l T

SK

HW

-55F

co

lum

n (6

0 ×

3.2c

m)

at a

flo

w r

ate

of 3

5ml/

h at

4°C

. T

he r

oman

nu

mer

als

I, 1

I, II

I an

d IV

and

arr

ows

refe

r to

the

mol

ecul

ar

wei

ght

stan

dard

s al

dola

se,

seru

m

albu

min

, ov

albu

min

and

cy

toch

rom

e c.

Labo

rato

ry

Expo

sed

~ Ill

Jv

Dig

esti

ve

Gla

nd

__ 2

80nm

, I

t"

" "t

~

I I

i 1.0

0.5

~

i 1.

o ~.

a: ~,.

~

0.5

~"

3 ~

~°f

~~'~

o

0.5 30

40

50

60

70

80

90

10

0 q

FRAC

TIO

N NU

MSE

R ~,

F

ig.

2.

Elu

tion

pro

file

of t

he c

ytos

olic

fra

ctio

n fr

om d

iges

tive

glan

d (h

epat

opan

crea

s) o

f bl

ue c

rabs

exp

osed

in

the

labo

rato

ry.

Con

dit

ion

s ar

e th

e sa

me

as i

n F

ig.

1.

T~


2.(:1

1.6

1.2

<

n- O 2.C ¢0 m

< 1.~

1 . 2

7 Digest ive Gland ~'~ I II III IV

i i' ' ................

. _ _ . A b s o r b a n c e . . . . ~Cd

o . . . . . o Cu ~ - ~ o Z n

Gill

/

3 0 4 0 50 6 0 7 0 8 0 J

0 " 2 0 9 l0

,2.4

2 .0

1.6

1: 1.2 .6

5 . 0 0

4.0

3.0 .6

Z.0 4

1.0 .2

F R A C T I O N N U M B E R

Fig. 3. Sephadex G-75 elution profiles of the cytosolic fractions from hepatopancreas and gills of blue crabs exposed to cadmium via food (dosed oysters) and water (100 ppb) for 14 days. Chromatographic separations were made on a 2.6 x 65cm column at a flow rate of 30ml/h. The eluant was 10mM phosphate buffer, pH8.0, at 4°C (Brouwer, unpublished data). The roman numerals l, II, IlI and IV and arrows refer to the molecular weight standards bovine serum albumin, ovalbumin, chymotrypsinogen and

ribonuclease A.

In the digestive gland there was a single protein peak at a molecular weight of about 10 000 that contained cadmium, copper and zinc. In the gills, however, the cadmium protein peak also contained a small amount of copper. These elution profiles agreed well with our earlier data for blue crabs exposed to cadmium from food or water for 14 days (Fig. 3). It appears, therefore, that dose rate (10 ppb for 40 days vs 100 ppb for 14 days) does not affect the distribution of metals on the metallothionein- like proteins in blue crabs. Since two different gel-filtration media were used, the similarities in the metal distributions on these proteins were reinforced. Also, in both sets of experiments the molecular weight estimates for the cadmium-binding (metallothionein-like) protein agreed

TA

BL

E 1

C

on

cen

trat

ion

s of T

race

Met

als

in th

e T

issu

es o

f B

lue

Cra

bs,

Cal

line

ctes

sap

idus

, C

olle

cted

fro

m t

he N

ewpo

rt R

iver

Est

uary

, N

ort

h C

aro

lin

a,

and

the

Hu

dso

n R

iver

Est

uary

, N

ew Y

ork

(All

val

ues

are

a m

ean

of

5 an

imal

s an

d co

nce

ntr

atio

ns

are

in/~

g/g

wet

wei

ght

of t

issu

e _+

SE

)

Loc

atio

n D

iges

tive

gla

nd

Gil

l M

uscl

e

Cd

Cu

Zn

N

i C

d C

u Z

n

Ni

Cd

Cu

Zn

N

i

Bea

ufor

t,

1.0

7.8

36.0

<

0.3

20

.0

7.2

0 5.

0 59

-0

NC

+

0.2

8

-+2

.1

-+8-

6 --

+

3.5

_+

0.8

0 _+

0.6

_+5-

6

Fo

un

dry

21

.0

64.0

39

.0

3.0

2.6

65.0

16

.0

1.4

0 22

.0

46.0

C

ove,

NY

_+

9.0

+1

2.0

_+

4-0

_+0.

7 +_

_1.0

+

13

.0

-+1.

0 +

0.8

0

-+4.

0 -+

11.

0

Hav

erst

raw

1

0.0

24

6.0

40

2.8

< 0

.6

76.0

15

.0

2.2

0 18

.0

47.0

B

ay,

NY

-+

1.8

-+76

.0

_+

4.0

_

+0

.6

--

_+

17

-0

_+

3.0

_

+0

.4

0 _+

5-0

_+ 1

0-0

1"6

_+0"

6

0"6

_+0"

1


well with the estimates made by Wiedow et al. (1982) for blue crabs collected from the Hudson River.

Cadmium, copper, zinc and nickel concentrations in the tissues of blue crabs collected from Beaufort and the two Hudson River locations showed significant differences in concentration depending upon site of collection (Table 1). Tissues of crabs collected from the Beaufort area were relatively low in copper and cadmium because there are no known significant inputs of these trace metals. The gills and digestive glands of Foundry Cove crabs were high in cadmium, but none was detectable in the backfin muscle. Such concentrations were undoubtedly related to high levels of cadmium in the sediments of Foundry Cove (50 000 mg/kg dry weight) (Kneip & Hazen, 1979). Only the digestive glands of Haverstraw Bay crabs were high in cadmium, but at about half the concentration found in the digestive gland of crabs from Foundry Cove. Our cadmium measurements agreed with those of Wiedow (1981), but his measurements did not include the other trace metals. All three tissues of crabs from both sites on the Hudson River had measurable nickel. Its presence was probably associated with the battery plant, because sediment concentrations were 2000 4800mg Ni/kg dry weight of sediment (Kneip & Hazen, 1979). Concentrations of copper in the digestive glands of crabs from both sites on the Hudson River were significantly higher than in crabs from Beaufort, and copper concentrations in crabs from Haverstraw Bay were substantially higher than in crabs from Foundry Cove. Differences between Haverstraw Bay and Foundry Cove crabs may be related to the steam electric station at Indian Point in the vicinity of Haverstraw Bay. Levels of copper in the gill tissue were difficult to compare, since variable amounts of hemocyanin retained in this tissue can influence the copper concentrations. Zinc concentrations in the digestive gland, gills and muscle of crabs from all three locations were remarkably similar.

The elution profiles of digestive glands and gills of crabs collected at Foundry Cove (Figs 4 and 5) provided a very good basis for comparison with the laboratory-exposed crabs (Figs 1 and 2). The agreement was excellent, with the gills having proteins that bound cadmium and a small amount of copper, and the digestive gland having proteins that bound copper, cadmium and zinc and that were of the appropriate molecular weight.

The Haverstraw Bay crabs (Figs 6 and 7) had elution profiles that were also very similar to the laboratory-exposed animals. In the gill tissue,


I II III tV F o u n d r y C o v e ~, ~. ~, GILL

2.0 f": e, I i~: . . . . ~ o $ "~ | ! _ _ 254

F i g . 4 .

A L 0.5

f 0.3

O < 0.1

0

. 3° I ~ 2.0

~ 1.0

0

~o. 3O

A I J l

I I

40 50 60 70 80 I I

90 100

FRACTION NUMBER

Elution profile of the cytosolic fraction of gills from blue crabs collected at Foundry Cove, NY. See Fig. 1 for conditions.

where the cadmium concentration was very low (Table 1), no identifiable cadmium-binding protein was observed, and neither zinc- nor copper- binding proteins were present in the fraction that corresponds to a molecular weight of 10 000. In the digestive glands, where there were high concentrations of" copper, a distinct copper-binding protein was demonstrated, but owing to the large quantities of copper in this tissue, copper was also associated with other lower molecular weight materials ( < 5000 Mr). Small amounts of cadmium were present as a peak eluting at about 10 000 M r.

The very close agreement between the zinc concentrations in the digestive glands, gills and muscles of blue crabs from all locations, and the


Foundry C o v e

I II I I I IV Digestive Gland

<z l l ; '~ ; " ~ ' '

~f~v /i b l q

Q. Q.

< 0

n,- w cu

0

1.0

0 .5

2.0

1.5

1.0 °:I 1.5

1.0

0 5 z N

....

3 0 4 0 50 6 0 7 0 8 0 I

9 0 100

Fig. 5. FRACTION NUMBER

Elution profile of the cytosolic fraction of hepatopancreas from blue crabs collected from Foundry Cove, NY. See Fig. 1 for conditions.

similar zinc distributions in all of the elution profiles, strongly suggest that zinc levels must be regulated extremely well. The close agreement in the digestive glands of the different groups of crabs may be fortuitous, but if not, then zinc metabolism must be controlled rigidly and be independent of geographical location. Also, the mechanism of this regulation must involve something other than a metallothionein-like

Hav

erst

raw

B

ay

Gil

ls

I It

III

IV

! ,

__,

254n

m

ol...

J,

"'L_

,

,

Q.

UJ

a.

0

5.0-

4.0

-

3.0

-

2.0

-

1.0

- oY

i

I I

I I

I 30

40

50

6

0

70

8

0

90

10

0

Hav

erst

raw

B

ay

i H

m

Jv

D

ige

sti

ve

G

lan

d

0 I

I ,-.

-'--..

..~--

.---~

- h

L I

J

"~ 3

.0

g ~c 2

.0

uJ

CL 1,

0

"~ o.5~

30

40

50

60

70

80

90

I

lOO

c3

ca.

5 5'

FR

AC

TIO

N

NU

MB

ER

Fig.

6.

Elu

tion

pro

file

of

the

cyto

soli

c fr

acti

on o

f gi

lls f

rom

bl

ue c

rabs

col

lect

ed a

t H

aver

stra

w

Bay

, N

Y.

See

Fig.

1 f

or

cond

itio

ns.

FR

AC

TIO

N

NU

MB

ER

Fig.

7.

Elu

tion

pro

file

of

the

cyto

soli

c fr

acti

on o

f di

gest

ive

glan

d (h

epat

opan

crea

s) f

rom

blu

e cr

abs

coll

ecte

d at

Hav

erst

raw

B

ay,

NY

. Se

e Fi

g. 1

for

con

diti

ons.


protein as a mediator, since only a small proportion of the total cytosolic zinc was bound to a metallothionein-like protein.

The fourth metal that we analyzed for was nickel, which was present at low concentrations in all tissues from crabs collected at Foundry Cove and Haverstraw Bay. In our elution profiles there was some suggestion of nickel binding to some components in the chromatographed material, but no nickel was bound to a protein of about 10 000 M r. Nickel metabolism should be investigated more fully to establish the types of molecules involved in the binding or transport of nickel in crabs.

Our investigations demonstrate that there is a good agreement between the patterns of trace metal partitioning in blue crabs exposed to cadmium in the laboratory and those exposed to elevated levels in the environment. While cadmium was the metal of prime interest, good agreement was observed between copper and zinc partitioning in the digestive glands and gills of the various groups of blue crabs. Such information is important if we are to understand the role of metal-binding (metallothionein-like) proteins in both the normal metabolism and trace metal detoxification processes in the blue crab and other marine crustaceans.

A C K N O W L E D G E M E N T S

The authors thank Dr Joseph O'Connor of the New York University Institute of Environmental Research, Tuxedo Park, NY, for his assistance in collecting the blue crabs from Foundry Cove and Haverstraw Bay. We also acknowledge the technical assistance given by Mr William J. Bowen III in analysis of the samples. This research is supported by a research grant to the NMFS Southeast Fisheries Center's Beaufort Laboratory and to the Duke University Marine Laboratory from the Ocean Assessment Division of the National Ocean Services, NOAA (Grant NA80-RA-D-00063), and partially by a Duke University Marine Biomedical Center grant (EOS 1908).

REFERENCES

Breteler, R. J. (Ed.) (in press). Chemical pollutants oj the Hudson-Raritan estuary. Battelle, New England Research Laboratory, Duxbury, Mass.

Engel, D. W. (1983). The intracellular partitioning of trace metals in marine


shellfish. In: Biological availability oJ'trace metals (Wildung, R. E. & Jenne, E.A. (Eds)). Elsevier, Amsterdam, 129-40.

Engel, D. W. & Fowler, B. A. (1977). Accumulation and biological effects of copper and cadmium on marine shellfish. In: Environmental eJ]ects of energy related activities on marine/estuarine ecosystems. Environmental Protection Agency, Washington, EPA-600/7-77/111, 45-56.

Evans, D. W. (1977). Exchange of manganese, iron, copper, and zinc between dissolved and particulate forms in the Newport River estuary, North Carolina. PhD dissertation, Oregon State University, Corvallis, Oreg., 218pp.

Kneip, T. J. & Hazen, R. E. (1979). Deposit and mobility of cadmium in a marsh-cove ecosystem and the relation to cadmium concentration in biota. Environ. Health Perspect. 28, 67-73.

Olafson, R. W., Sim, R. G. & Boto, K. G. (1979). Isolation and chemical characterization of the heavy metal-binding protein metallothionein from marine invertebrates. Comp. Biochem. Physiol. 62B, 407-16.

Overnell, J. & Trewhella, E. (1979). Evidence for the natural occurrence of (cadmium, copper)-metallothionein in the crab Cancer pagurus. Comp. Biochem. Physiol. 64C, 69 76.

Ridlington, J. W. & Fowler, B. A. (1979). Isolation and partial characterization of a cadmium-binding protein from the American oyster (Crassostrea virginica). Chem.-Biol. Interact. 25, 127-38.

Ridlington, J. W., Chapman, D. C., Goeger, D. E. & Whanger, P. D. (1981). Metallothionein and Cu-chelatin: characterization of metal-binding proteins from tissues of four marine animals. Comp. Biochem. Physiol. 70B, 93-104.

Roesijadi, G. (1981). The significance of low molecular weight, metallothionein- like proteins in marine invertebrates: current status. Mar. Environ. Res. 4, 167 79.

Wiedow, M. A. (1981). Distribution and binding of cadmium in the blue crab (Callinectes sapidus): implications in human health. PhD thesis, New York University, 69 pp.

Wiedow, M. A., Kneip, T. J. & Garte, S. J. (1982). Cadmium-binding proteins from blue crabs (Callinectes sapidus) environmentally exposed to cadmium. Environ. Res. 28, 164 70.

Documents

Cadmium-binding proteins in the blue crab, callinectes sapidus: laboratory-field comparison