HELGOL~NDER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters . 33, 278-291 (1980)

Mixed function oxygenases and xenobiot ic

detoxicat ion/toxicat ion systems in bivalve molluscs

M. N. Moore, D. R. Livingstone, P. Donkin, B. L. Bayne, J. Widdows & D. M. Lowe

Natural l~nvironment Research Council, Institute for Marine l~nvironmental Research; Prospect Place, The Hoe, Plymouth PL1 3DH, England

ABSTRACT: Components of a xenobiotic detoxication/toxication system involving mixed function oxygenases are present in h4ytilus edulis. Our paper critically reviews the recent l i terature on this topic which reported the apparen t absence of such a system in bivalve molluscs and attempts to reconcile this v iewpoint with our own findings on NADPH neotetrazol ium reductase, glucose-6- phospha te dehydrogenase, aldrin epoxidat ion and other reports of the presence of mixed function oxygenases. New exper imenta l data are presented which indicate that some e lements of the detoxicatio~/toxication system in M. edulis can be induced by aromatic hydrocarbons der ived from crude oil. This includes a brief review of the results of long- term exper iments in which mussels were exposed to low concentrat ions of the water accommodated fraction of North Sea crude oil (7.7-68/~g 1-1} in which genera l stress responses such as reduced physiological scope for growth, cytotoxic damage to lysosomal integri ty and cellular damage are considered as characteristics of the genera l stress syndrome induced by the toxic action of the xenobiotics. In addition, induct ion in the blood cells of microsomal NADPH neotet razol ium reductase {associated wi th mixed function oxygenases) and the NADPH genera t ing enzyme glucose-6-phosphate dehydrogenase are con- s idered to be specific biological responses to the presence of aromatic hydrocarbons. The conse- quences of this detoxication/toxication system for Mytilus edulls are discussed in terms of the formation of toxic electrophilic in termediate metabol i tes which are highly reactive and can combine with DNA, RNA and proteins wi th subsequent damage to these cellular constituents. Implications for neoplasms associated wi th the blood cells are also discussed. Finally, in view of the increased use of mussel species in pol lutant moni tor ing programmes, the induct ion p h e n o m e n o n which is associated wi th microsomal enzymes in the blood cells is considered as a possible tool for the detect ion of the biological effects of envi ronmenta l contaminat ion by low concentrat ions of certain groups of organic xenobiotics.

I N T R O D U C T I O N

C y t o c h r o m e P - 4 5 0 - d e p e n d e n t m i x e d f u n c t i o n o x y g e n a s e s ( m o n o o x y g e n a s e s ) a re

i n v o l v e d i n t h e t r a n s f o r m a t i o n , to p o l a r m e t a b o l i t e s , of a w i d e r a n g e of o r g a n i c c o m -

p o u n d s i n c l u d i n g m a n y f o r e i g n n o n - b i o l o g i c a l c o m p o u n d s or x e n o b i o t i c s (Kr i ege r 8,

W i l k i n s o n , 1969; C o n n e y & B u m s , 1972; B e n d e t el., 1977}. T h e s e e n z y m e s a r e f re-

q u e n t l y a s s o c i a t e d w i t h t h e e n d o p l a s m i c r e t i c u l u m ( m i c r o s o m e s ) a n d t h e m e m b r a n e s of

t h e n u c l e a r e n v e l o p e ( K h a n d w a l a & K as pa r , 1973); t h e y r e q u i r e N A D P H a n d o x y g e n .

�9 Biologische Anstal t Helgoland 0017-9957/80/0033/0278/$ 02.00

Detoxication/toxication systems in bivalves 279

This system can function as a mechanism for the detoxication of organic environmental contaminants; however, in the course of such transformations reactive electrophilic metabolites can be formed (Kuroki et al., 1971-72~ Daly et al., 1972; Jerina & Daly, 1974~ Jollow et al., 1974~ Sims & Grover, 1974), some of which are carcinogens, mutagens or other cytotoxic agents. The mixed function oxygenases must therefore be viewed as components of a detoxication/toxication system, as some of the metabolites may be more biologically damaging than the parent compound.

A number of previous investigators have argued that bivalve molluscs do not have the capability to metabolise organic xenobiotics (Carlson, 1972~ Lee et al., 1972~ Payne, 1977~ Vandermeulen & Penrose, 1978) and this argument has been used as a partial explanation for the persistence of hydrocarbons within the tissues of bivalves exposed to contamination (Lee et al., 1972). However, recent evidence suggests that xenobiotic detoxication systems are present in bivalves although the levels of activity are low in comparison with fish and mammals. Bend et al. (1977) have demonstrated the presence of epoxide hydrase in the digestive gland of the blue mussel, Mytilus edulls, while the epoxidation of aldrin to dieldrin by a mixed function oxygenase has been reported in Anodonta sp. (Khan et al., 1972), in Mytilus californianus (Gee et al., 1979; Krieger et al., 1979) and in M. edulis (Bayne et al., 1979; Widdows et al., 1980a). Trautman et al. (1979) have also reported the presence of mixed function oxygenase-mediated o-demethylation of p-nitroanisole in whole-body microsomal preparations from M. californianus, while Krieger et al. (1979) have shown that this mussel can also hydroxylate antipyrine (2,3- dimethyl-1-phenyl-2-pyrazolin-5-one) in vivo. The hydroxylation of benzo(a)pyrene by a mixed function oxygenase has also been reported to occur in M. edulis (Stegeman, 1980)~ Payne, personal communication).

Evidence of an association of the detoxication system with particular tissues and cells in hi. edulis has been indicated from cytochemical investigations of the distribution of NADPH neotetrazolium reductase (Bayne et al., 1979~ Moore, 1979, 1980; Widdows et al., 1980a), which appears to represent the oxidation of NADPH by the microsomal respiratory chain terminating in the mixed function oxygenase (Koudstaal & Hardonk, 1969, 1972~ Richards, 19731 Hardonk & Koudstaal, 1976; Chayen, 1978; Lindner & Beyhl, 1978). Previous results show that NADPH neotetrazolium reductase is particularly active in the blood cells (haemocytes) of hz/. edulis, although there is also some activity associated with both the intestinal epithelium and developing oocytes (Moore, 1979). This evidence of comparatively high activity in the blood cells is complemented by correspondingly high levels of mixed function oxygenase (aldrin epoxidation) in these cells (Widdows et al., 1980a).

The occurrence of enzymic-induction of some components of the microsomal detoxi- cation system (see review by Conney & Bums, 1972) is evident from investigations of the effects of phenobarbital, anthracene, phenanthrene, 2,3-dimethylnaphthalene and the water accomodated fraction (WAF) of crude oil which all cause a stimulation of NADPH neotetrazolium reductase activity (Bayne et al., 1979; Moore, 1979, 1980), as well as the NADPH generating enzymes (WAP only tested) glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase (Bayne et al., 1979).

As stated above, the detoXication system can facilitate the formation of reactive intermediate metabolites which have a potential for damage to cellular macromolecules including DNA (Gillette et al., 1978; Lesko et al., 1978). In this context neoplastic blood

280 M . N . Moore et al.

cells have b e e n r epor t ed from a n u m b e r of b iva lves i nc lud ing M. edulis (Farley, 1969a, b; Fa r l ey & Sparks , 1970; Barry & Yevich, 1972~ Mix, 1975; Mix et al., 1977) whi le Lowe & Moore (1978) have demons t r a t ed that neop las t i c mussels , from a popu la t ion nea r Plymouth, were na tu ra l ly exposed to a r ange of po lycyc l ic a romat ic hydrocarbons . However , the ev idence for any associa t ion b e t w e e n the p resence of the de tox ica t ion / toxicat ion sys tem in the b lood cel ls and the occurrence of haemocy t i c neop la s i a is pu re ly c i rcumstant ia l and wi thout expe r imen t a l induc t ion of this la t te r condi t ion must r e m a i n specula t ive .

In this p a p e r we repor t on expe r imen ta l l y i n d u c e d effects of the W A F of North Sea crude oil and 2 ,3 -d ime thy lnaph tha l ene on m i x e d funct ion oxygenase , NADPH neote t ra- zo l ium reduc tase and NADPH gene ra t i ng enzymes in M. edulis.

MATERIALS AND METHODS

E x p e r i m e n t a l t r e a t m e n t of m u s s e l s w i t h h y d r o c a r b o n s

Musse ls (Mytilus edulis), 50 .60 nun in shel l length, were col lec ted from the es tuary of the River Erme (Devon} and he ld at the seasona l a m b i e n t t empe ra tu r e in a sys tem of rec i rcu la t ing seawater . Al l musse ls were fed an a lga l d ie t of Phaeodactylum tricornutum and Isochrysis galbana at a concent ra t ion of 4000-6000 cel ls m1-1.

Musse ls were cont inuous ly exposed in a f low- through sys tem to low concentra t ions of the wa te r a c c o m m o d a t e d fract ion (WAF) of North Sea Crude Oil. In this p a p e r we p resen t resul ts form three exper iments : Exper imen t h N o v e m b e r 1977, 12 #g total hydrocarbons 1-1 (__ 2.2 S.E.); Exper imen t Ih Apr i l 1978, 7.7 #g 1-1 (__ 0.7 S.E.) and 68/2g 1-1 (___ 9 S.E.); Exper iment IIh Oc tober 1978-Apr i l 1979, 30 ka:j 1-1 ( - 4.5 S.E.). A stock of wa te r a c c o m o d a t e d hydroca rbon (4.2 mg 1-1) was p r e p a r e d dai ly, by gen t ly mix ing 20 1 of s eawa te r and 250 ml of crude oil on a magne t i c st i rrer for a pe r iod of 18 h and then l eav ing und i s tu rbed for a further 6 h.

In Exper imen t I, the me thod of dos ing and d i lu t ing the s tock W A F differed from that of Exper iments II and HI. Exper imen t I a ch i eved an exposure concent ra t ion of 12/~g 1-1 by p u m p i n g 20 1 of s tock pe r day into the s eawa te r aqua r ium (volume 3000 1). Dur ing this expe r imen t the seawa te r was ne i the r a e r a t ed nor f i l tered. In Exper iments II and III the s tock W A F was p u m p e d (13 ml min -I) into the s t ream of inf lowing seawa te r before en te r ing the t ank con ta in ing the expe r imen t a l an imals . The r equ i r ed exposure concen- t ra t ion was therefore d e t e r m i n e d by the f low rate of incoming seawater . Under these expe r imen t a l condi t ions the hydroca rbon levels in the total s eawa te r sys tem were m a i n t a i n e d at b a c k g r o u n d concentra t ions by m e a n s of ae ra t ion and fil tration. Control an imals were he ld in a s epa ra t e sys tem of rec i rcu la t ing seawater .

The hydroca rbon concentra t ions in the stock, e xpe r ime n t a l and control t anks were mon i to red twice a week . Fo l lowing ext rac t ion into hexane , e i ther by m e a n s of shak ing or s team dis t i l la t ion, the hydrocarbons were m e a s u r e d in terms of n a p h t h a l e n e equ iva len t s at 220 nm. Add i t i ona l wa te r samples were a n a l y s e d by gas ch roma tog raphy in order to der ive a convers ion factor from n a p h t h a l e n e equ iva len t s to total hydrocarbons .

In a fur ther ser ies of expe r imen t s musse ls were in jec ted wi th e i ther W A F (7.6 m g 1-1) as p r e p a r e d above or wi th 2 ,3 -d ime thy lnaph tha l ene (July, 1979). Inject ions (100/A/

Detoxication/toxication systems in bivalves 281

mussel) were made into the posterior adductor muscle using a 26-gauge hypodermic needle for WAF together with a mill ipore-fi l tered seawater (MFSW) vehicle control, and a 23-gauge needle for 2,3-dimethylnaphthalene (10-2M) dissolved in medicinal l iquid paraffin with a l iquid paraffin vehicle control. Tissues were sampled for enzyme assays after 24 h at 15 ~ the seasonal ambient temperature.

A s s a y for m i x e d func t ion o x y g e n a s e

Blood (between 0.5 and 2 ml per mussel) was removed from the posterior adductor muscle by means of a syringe and bulked, to a total volume of 30 ml, in a polycarbonate centrifuge tube. Blood cells were sedimented by cenirifugation at 500 g for 10 min then transferred to a glass Potter-Elvehjem homogeniser vessel in a total volume of 6 ml of 0.1 M HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulphonic acid} (pH 7.6} buffer containing NaC1 (3 %, 0.514 M) and 10 mM MgSO4. After addit ion of an ethanol solution of phenylmethylsulphonylf luoride (PMSF) (2 ~l of solution m1-1 buffer to a final PMSF concentration of 0.1 raM) the cells were homogenised by 10 passes of a motor-driven teflon pestle.

Mantle tissue and digestive glands were dissected from mussels not previously used for blood sampling and homogenised essentially as for blood, but using 20 ml of buffer g-1 wet weight of tissue. For each experimental treatment, either 5 or 6 animals were assayed individually. Homogenates were centrifuged at 10,000 g for 30 min and clear supematant was removed for assay. Blood, dissected tissue and homogenates were mainta ined at or below 4 ~ throughout.

Aldrin epoxidase activity was determined in incubations of the following composi- tion: 0.1 M HEPES buffer (pH ?.6)~ MgSO4, 10 mM~ NaCll 0.51 M~ NADPH 0.3 mM~ glucose-6-phosphate, 1.8 re_M; tissue homogenate 0.8 ml (digestive gland and mantle) or 3.8 ml (blood) and aldrin, 0.23 ~rnole, added in 10 Ill of methanol. Total volumes were adjusted to 4 ml with buffer and the incubations carried out aerobically at 25 ~ for 1 h with frequent shaking. Blanks were run without cofactors, with homogenate replaced by buffer, and with all components but s topped at zero time.

The incubations were stopped by extraction with 4 ml of 15 % (v/v) diethyl ether in hexane followed by cooling. Extraction was completed with a further two 4-ml volumes of ether/hexane, the bu lked extracts concentrated to 2 ml and analysed for dieldrin by electron capture detector gas chromatography.

Homogenate protein concentrations were assayed by the method of Lowry et al. (1951} relative to a bovine serum albumen (fraction V) standard calibration curve prepared with appropriate dilutions of homogenising buffer.

Q u a n t i t a t i v e c y t o c h e m i c a l a s s a y for N A D P H n e o t e t r a z o l i u m r e d u c t a s e

A small piece (approx. 5 mm 3) of the visceral mass from experimental mussels was rapidly excised, frozen in hexane (- 70 ~ and sectioned (10 into) in a Bright Cryostat at a cabinet temperature of - 25 to - 30 ~ with a dry-ice cooled knife. NADPH neotetrazo- lium reductase was demonstrated in unfixed sections using a modification of the method described by Altman (1972} which employed 100 mM H~.PES buffer (pH 8.0) containing 20 mM MgC12, 20 % PVA and 5 mM neotetrazolium chloride (Sigma). Incubation was for

282 M.N. Moore et al.

30 min in a nitrogen atmosphere at 37 ~ Control sections omitted NADPH from the incubation medium. Microdensitometric measurements were made on individual blood cells in sections stained for NADPH ne0tetrazolium reductase (10 readings/section) using a Vickers M 85 scanning integrating microdensitometer at a wavelength of 585 nm (Butcher and Altman, 1973; slit width setting -- 60; measuring spot -- 0.5 fan diameter). The data were analysed using the Mann-Whitney U-test between control and experi- mental groups and a probabil i ty value of less than 0.05 was accepted as significant.

A s s a y s for g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e

On each sampling occasion, individual mussels were taken and a sample of blood removed from the posterior adductor muscle by hypodermic syringe. The digestive gland was excised, damp-dried, weighed and placed on ice. All procedures were carried out at 4 ~ The blood samples were centrifuged at 1000 g for 10 min and the blood cells resuspended in the same volume of sterilised seawater. After a second spin, the blood cells were resuspended in the same volume 'of 10 mM tris-HC1 pH 7.7 containing 1 mM EDTA (ethylenediamine tetra-acetic acid) and 1 mM DTT (dithiothreitol) and sonicated for three 5-s periods using a MSE sonicator operating at an ampli tude of 18 #m. The sonicated samples were assayed for glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49- G6PDH) as described in Bayne et al. 11979), and for protein either by the direct spectrophotometric method of Groves et al. (1968), or by the method of Lowry et al. (1951).

Excised digestive g lands were gently homogenised in approximately 15 volumes (W : V) of 0.8 M sucrose containing 10 mM tris-HC1 pH 7.7, 1 mM EDTA and i mM DTT using an ultra-turrax homogeniser, The homogenates were centrifuged at 40,000 g for 30 min and the resulting supernatants assayed for G6PDH as above. In later experiments (see Table 7), this supernatant was further centrifuged at 100,000 g for 90 min to yield a high-spin supernatant (cytosolic G6PDH) and a microsomal pel let which was resuspen- ded in a minimum volume of homogenising buffer (sucrose omitted) (microsomal G6PDH). The data were analysed by one-way analysis of variance between two groups of values or for several group values over time. A probabil i ty value of less than 0.1 was accepted as significant.

RESULTS AND DISCUSSION

M i x e d func t ion o x y g e n a s e

Aldrin epoxidation has been demonstrated in 10,000 g supernatants (generally denoted "post mitochondrial" supernatant, PMS) from three tissues of M. edulis (Table 1). Dieldrin production per incubation increased l inearly as PMS concentration was increased up to 0.8 mg of protein m1-1 of incubate, and activity was totally destroyed by heat (100 ~ for 3 min) indicating an enzymic mechanism. Omission of NADPH gene- rally reduced epoxidase activity to the level of the blanks, though occasionally up to 20 0/0 of the activity of the complete system was present, probably mediated by endoge- nous cofactors. This NADPH requirement provides evidence for a mixed function oxygenase mechanism for the metabolism of aldrin.

Detoxica t ion / tox ica t ion sys tems in b iva lves 283

Table 1. Epoxidation of aldrin by 10,000 g supernatants derived from M. edulls tissues*

Tissue Rate of dieldrin formation (pmole h -I mg protein -1)

Mean___ SE, n = 6

Mantle 27.7 _+ 5.7 Digestive gland 2.0 _ 0.5 Blood cells '* 54.9

* Mussels sampled during May 1978 * * Single incubatiom blood pooled from 30 animals

Rates of a ldr in epox ida t ion in man t l e t issue (wet we igh t basis ; Table 2} were s imi lar to those r epor t ed by Khan et al. (1972) for d iges t ive g l a n d of the f reshwater musse l Anodonta sp. (3.07 nmole h - lg -1) and by Wil l is & A dd i son (1974} for b i p h e n y l hydroxy la - t ion by h o m o g e n a t e s of whole M. edulis {6.3 • 4.6 S.D. nmole h-lg-1). Expressed on a we t we igh t basis, the m e a n ra te for the resul ts for man t l e t issue g iven in Tab le 1 was 1.67 • 0.23 (mean • SE, n = 6} nmole h - lg -1. Expressed on a p ro te in bas is (as in Tab le 1) this rate was b e l o w that r epo r t ed for a ldr in epox ida t ion (60-300 pmole h-~mg -1) by Kr ieger et al. (1979} in var ious t issues of Myti lus callfornianus; comparab l e ra tes for rat l iver were 6,000 p m o l e h-~mg -1 (Krieger e t al., 1979}. At the t ime of the assay p r e s e n t e d in Tab le 1 (May}, epox ida t ion ra tes in mant le were g rea te r t han in d iges t ive g land, a l though the resul ts for d iges t ive g l and must be i n t e rp re t ed wi th caut ion {see below}. Blood cells showed the h ighes t ac t iv i ty and if these rates were c o m p a r e d us ing an e s t ima ted b lood cel l we t we igh t (see Widdows et al., 1980a), b lood cel ls were scored as the most act ive t issue. Fur ther s tudies (Donkin, u n p u b l i s h e d data} have shown that a l though b lood cel l epox idase act ivi ty can be high, this is not the case for al l groups of an ima l s tested. The reason for this va r i ab i l i t y is not ye t clear.

Despi te repor ts that a ld r in epox idase act ivi ty is i nduc ib le in fish by pe t ro l eum pol lu t ion (Burns, 1976} and 3 -me thy lcho lan th rene (Addison et al., 1978), in jec t ion of W A F or 2 ,3 -d ime thy lnaph tha l ene fa i led s igni f icant ly to inc rease epox idase act ivi ty in man t l e s of M. edulls (Table 2). Var iab i l i ty was cons iderab le , however , due poss ib ly to the s amp le s b e i n g a mix ture of pre- and pos t - spa w n ing spec imens of bo th sexes.

Table 2. Epoxidation of aldrin by 10,000 g mantle tissue* supematants derived from M. edulis injected with WAF and 2.3-dimethylnaphthalene sampled 24 h after injection

Treatment

Rate of dieldrin formation (nmole h -I g wet wt. -1)

Mean • SE, n = 5 Control Experimental

Injection of 100/~1 of WAF (7.63 mg 1-1) Injection of 100/~1 of 2,3-dimethylnaphthalene (10 -2 M)

* Digestive glands were also assayed~ see text

2.77 -- 0.45 2.88 • 0.38 2.40 • 0.59 2.56 • 1.00

284 M . N . Moore et al.

Table 3. Responses of NADPH neotetrazolium reductase activity in the blood cells of M. edulis exposed to WAP of North Sea crude oil

l~.xperiment Time (days)

Relative absorbance of NADPH neotetrazolium reductase activity

(machine units of absorbance) Mean • SE, n = 5

Control t~xperimental

I (12/~9 l-l) 7 18.23 • 2.00* 33.84 _ 3.05 16.21 _ 2.13" 33.09 • 2.37

14 32.06 • 3.23 30.32 • 3.07

35 21.30 • 1.94 35.63 • 2.29 23.94 • 2.19 42.18 • 3.91

II (7.7/~j 1 -I) 3 12.79 • 1.56 20.80 • 2.50 II (68/~j i -I) 3 28.41 • 2.82

* Duplicate samples in Experiment I

Digest ive g lands gave even more var iable results. Animals in jec ted wi th 2,3-dimethyl- naph tha l ene showed no activity whereas those in jec ted with WAF formed die ldr in at the rate of 0.96 _- 0.44 compared to control 0.19 ___ 0.03 (mean ___ SIR, n = 4; nInole h-lg-1). These results were of l imited value, however, since digest ive g land PMS conta ined const i tuents which, even w h e n di luted 20 times, could completely inh ib i t mant le activity. Krieger et al. (1979) also refer to var iable levels of inhibi tors in hi. californianus viscera.

The presence of epoxidase activity in blood cells further complicates the interpreta- t ion of results from the mant le and digest ive gland, since number s of blood cells in these tissues are var iable (Lowe & Moore, unpub l i she d observations).

N A D P H n e o t e t r a z o l i u m r e d u c t a s e

NADPH neote t razol ium reductase was pr inc ipal ly localised in the blood cells of Mytilus as found in previous exper iments (Bayne et al., 1979; Moore, 1979}. Microdensi- tometrical ly de te rmined activity from ind iv idua l blood cells showed that there was a s ignif icant increase after 7, 14 and 35 days of exposure to water accommodated fraction {Experiment I, Table 3). This induc t ion was consistent in replicate samples and constitu- ted almost a doub l ing of activity in blood cells from control mussels (Table 3). These results have received further confirmation from evidence of similar induc t ion of this enzyme in several other exper iments with water accommodated fraction (see Experi- men t II, Table 3).

In ject ion of exper imenta l mussels wi th WAF resul ted in s ignif icant induc t ion of NADPH neote t razol ium reductase after 24 h (2 repl icate experiments ; Table 4). Similar inject ions of 2 ,3-d imethylnaphtha lene also s t imulated an increase in activity after 24 h (Table 4).

A previous exper iment in which 2 ,3-d imethylnaphtha lene was in jec ted showed a

Detoxica t ion / tox ica t ion systems in b iva lves 285

Table 4. Responses of NADPH neotetrazolium reductase activity in the blood cells of M. edulis injected with WAF and 2,3-dimethylnaphthalene; sampled 24 h after injection

Treatment

Relative absorbance of NADPH neotetrazolium reductase activity

(machine units of absorbance) M e a n • n = 5

Control Experimental

Injection of 100/,1 of WAF (7.63 mg 1-1)

Injection of 100/11 of 2,3-dimethylnaphthalene (10 .2 M)

* Results from duplicate experiments

19.62 +__ 1.23" 24.62 ___ 0.48 18.39 • 0.49* 24.32 __ 0.84 17.81 • 0.49 23.53 ___ 0.76

much grea te r s t imula t ion of NADPH neo te t r azo l ium reduc ta se (Moore, 1979). However , this expe r imen t was car r ied out dur ing the la te win te r (Februa ry -March) before the s p a w n i n g per iod , whi l e the resul ts de sc r ibed above were ob ta ined after s p a w n i n g and dur ing the sexua l res t ing stage. The m i x e d funct ion oxygenase sys tem is invo lved in s teroid me tabo l i sm (see r ev iew by Conney & Burns, 1972) and this seasona l va r ia t ion in the induc t ion response of NADPH neo te t r azo l ium reduc tase in M. edu l i s m a y be in f luenced by the reproduc t ive cycle and its hormona l control.

G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e

An increase in G6PDH act ivi ty of the c o m b i n e d gi l l and k i d n e y t issues wi th exposure of h4. edu l i s to WAF was de t ec t ed in ea r l i e r exper iments , a l though ind iv idua l va r i ab i l i t y was h igh (see Bayne et al., 1979). However , fo l lowing the iden t i f ica t ion of the b lood cel ls as b e i n g pa r t i cu la r ly ac t ive for NADPH neo te t razo l ium reduc tase (Bayne et al., 1979; Moore, 1979, 1980); inves t iga t ions were concen t ra t ed on the b lood cel ls and on the d iges t ive g l and (a t issue r ich in b lood cel ls - see Moore, 1979).

The specif ic ac t iv i ty of b lood cel l G6PDH was h ighe r than the control leve ls du r ing the first th ree days of exposure of musse ls to W A F (Table 5). It was s igni f icant ly h i g h e r 24 h after the in jec t ion of 2 ,3 -d ime thy lnaph tha l ene but not after the in jec t ion of W A F (Table 5). Total b lood cel l G6PDH activity, wh ich reflects the n u m b e r of b lood cel ls p resen t in the h a e m o l y m p h , showed cons ide rab le va r i ab i l i t y b e t w e e n expe r imen t s and b e t w e e n i nd iv idua l musse l s (Table 5). This va r i ab i l i ty p r o b a b l y ar ises b e c a u s e the b lood cel ls mig ra te into and out of the t issues and at any m o m e n t in t ime a l a rge and va r i ab le fract ion m a y be p resen t in the t issues and absen t from the h a e m o l y m p h (Lowe & Moore, u n p u b l i s h e d observat ions) . However , cons ider ing the poo l ed da ta for the first th ree days of exposu re of musse l s to WAF, an increase in total b lood cel l G6PDH act ivi ty was observed. Diges t ive g l a n d G6PDH ( 'pos t -40,000 g supe rna tan t " - c o m b i n e d cytosol ic and mic rosomal activity) d id not inc rease wi th the exposure of musse ls to W A F excep t in one expe r imen t wi th long- te rm exposure (see Exper imen t IIl, Tab le 6). Control va lues va r i ed wi th the t ime of the yea r (Table 6) re f lec t ing the k n o w n seasona l pa t t e rn of change in G6PDH act iv i ty (Si lva-Pando et al., 1978; Head , 1979; Livingstone, 1980). In contras t to the exposure exper iments , bo th cytosol ic and microsomal G6PDH inc rea sed

2 8 6 M . N . M o o r e e t a l .

Tab l e 5. R e s p o n s e s of g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ac t iv i ty in t he b lood cel ls of M. edulis to W A F of N o A h Sea c rude oil a n d 2 , 3 - d i m e t h y l n a p h t h a l e n e

T r e a t m e n t T i m e Specif ic act ivi ty* Tota l act ivi ty* * (days) Cont ro l E x p e r i m e n t a l Cont ro l E x p e r i m e n t a l

E x p o s u r e to W A F (68 fig 1-1) 0.25 0.099 (Expe r imen t II) +

In jec t ion of 100 ~1 of W A F (7.63 m g 1-1) ++ In jec t ion of 100 #1 of 2,3- d i m e t h y l n a p h t b a l e n e (10 -2 M) ++

_ 0.014 0.126 • 0.017 6.81 _ 1.46 8.30 _ 1.28 (8)*** (8)

1.25 0.059 _ 0.012 0.128 _ 0.023 4.32 • 0.73 9.87 ___ 1.85 (8) (8)

3 0.090 • 0.012 0.164 _ 0.041 8.71 • 2.96 12.33 • 3.75 (8) (8)

7 0.126 • 0.019 0.126 • 0.016 6.04 • 1.67 7.02 • 1.02 (8) (8)

1 0.047 • 0.009 0.097 _ 0.029 12.36 • 3.86 13.59 _ 3.12 (6) (6)

1 0.058 • 0.012 0.102 • 0.017 20.74 • 4.98 20.69 • 3.84 (11) (11)

* /~moles m i n -1 m g p ro t e i n -1 ( m e a n + SE) * * n m o l e s m i n -1 ml b lood -1 ( m e a n _+ SE) * * * N u m b e r of i n d i v i d u a l s p e r s a m p l e + Pro te in m e a s u r e d by t he m e t h o d of Groves et al. (1968} ++ Pro te in m e a s u r e d by t he m e t h o d of Lowry et al. (1951)

Tab l e 6. R e s p o n s e s of g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ac t iv i ty of t he d ige s t i ve g l a n d of M. edulis e x p o s e d to W A F of Nor th Sea c rude oil

E x p e r i m e n t L e n g t h of e x p o s u r e

(days)

Specif ic act ivi ty* (~moles ra in -1 g we t wt -1)

M e a n _ S.E. Cont ro l E x p e r i m e n t a l

I (12 fig 1-I; Nov. 1977)

II (68 ~g 1-1 Apr. 1978)

III (30 #g 1-1; Feb. 1979)

7 2.13 + 0.47 1.40 + 0.11 (4)** (4)

22 2.10 + 0.19 1.70 • 0.15 (4) (4)

0.25 1.89 + 0.21 1.78 + 0.17 (8) (8)

1.25 1.25 + 0.16 1.02 + 0.17 (8) (8)

3 2.03 • 0 . I0 1.87 • 0 . i i (8) (8)

7 1.75 + 0.19 1.65 + 0.18 (8) (8)

210 1.16 + 0.06 1.41 + 0.09 (6) (6)

* G 6 P D H act iv i ty w a s m e a s u r e d in pos t -40 ,000 g s u p e r n a t a n t a n d r e p r e s e n t s t he s u m of cytosol ic a n d m i c r o s o m a l ac t iv i t ies

* * N u m b e r of i n d i v i d u a l s pe r s a m p l e

Detoxica t ion / tox ica t ion sys tems in b iva lves 287

Table 7. Responses of cytosolic and microsomal glucose-6-phosphate dehydrogenase activity of the digestive gland of M. edulis injected with WAF and 2,3-dimethylnaphthalene; sampled 24 h after

injections

Treatment Cytosolic* Microsomal * Control Experimental Control Experimental

Injection of 100 ~l of WAF (7.63 mg 1 -z) Injection of 100 ~1 of 2,3-dimethylnaphthalene (10 -2 M)

1.582 + 0.138 1.915 + 0.153 0.059 +_ 0.005 0.075 + 0.007 (11)** (ii) (11) (11)

1.448 +0.158 2.240 + 0.069 0.043 + 0.004 0.096 + 0.018 (ii) (II) (11) (II)

* Specific activities of both fractions in/~moles min -1 g wet wt -1 Mean __+ SE * * Number of individuals per sample

in act ivi ty fo l lowing an in jec t ion of 2 ,3 -d ime thy lnaph tha l ene whi le a s imilar , bu t reduced , effect was seen for the microsomal G6PDH fol lowing an in jec t ion of W A F (Table 7).

It is we l l e s t ab l i shed for m a m m a l i a n t issues that t r ea tment wi th such compounds as p h e n o b a r b i t a l l e ads to an inc rease in G6PDH act ivi ty (Altman, 1972). The resul ts are less def in i t ive for the effects of po lycyc l ic hydrocarbons wi th both inc reases (Koudstaal & Hardonk, 1972) and no change (Bresnick & Yang, 1964) in G6PDH act ivi ty b e i n g recorded. These d i f ferences have p robab ly a r i sen b e c a u s e the different t echn iques u s e d e i ther measu re cytosolic or microsomal act ivi t ies or both, and it is now rea l i s ed that the two G6PDH's a re gene t i ca l ly dis t inct enzymes , at leas t in ver tebra tes , wi th different p roper t i e s (see S t e g e m a n & Klotz, 1979). Cytosol ic G6PDH functions eff ic ient ly only wi th g lucose -6 -phospha te as substrate , wh i l e the microsomal G6PDH wi l l also u t i l i se ga l ac to se -6 -phospha t e and g lucose and is p r o b a b l y iden t i ca l wi th the enzyme formerly k n o w n as g lucose d e h y d r o g e n a s e (E.C. 1.1.1.47) (see Hori & Takahash i , 1974). The microsomal enzyme has b e e n r e t e r m e d hexose -6 -phospha t e d e h y d r o g e n a s e and has b e e n s u g g e s t e d as b e i n g impor tan t in p rov id ing r educ ing equ iva len t s for the mixed - funct ion oxygenase reac t ions (Kimura et al., 1979; S t e g e m a n & Klotz, 1979). By contrast, the cytosol ic G6PDH has a major role in the pen tose phospha te pa thway . The resul ts wi th M. edulis, therefore, ind ica te that changes in G6PDH act ivi t ies are par t of the response to a romat ic hydrocarbons bu t r e sea rch is r equ i r ed to resolve the funct ions of the two enzymes , pa r t i cu la r ly in the context of seasona l var ia t ions in act ivi ty and sex di f ferences (see Livingstone, 1980).

I n d u c t i o n of t h e d e t o x i c a t i o n s y s t e m as a s p e c i f i c r e s p o n s e to o r g a n i c x e n o b i o t i c s

The s t imula t ion of ac t iv i ty of m i x e d funct ion oxygenases and assoc ia ted microsomal enzymes b y a w ide va r i e ty of organic compounds has b e e n r ecogn i sed in m a m m a l s and fish for many years (see r ev iew by Conney & Bums, 1972; Payne & Penrose, 1975; S t e g e m a n & Sabo, 1976; Bend et al., 1977). A l though we have no def in i t ive ev idence of induc t ion of m i x e d funct ion oxygenase s in b iva lves , there is c lear ly an e n h a n c e m e n t of NADPH neo te t r azo l ium reductase , g lucose -6 -phospha te d e h y d r o g e n a s e and microso-

288 M.N. Moore et al.

mal hexose-6-phosphate dehydrogenase following experimental treatment with injected aromatic hydrocarbons and environmentally realistic levels of WAF. Accordingly, induc- tion of these systems in M. edulis and possibly other bivalves may have some potential for the monitoring of biological responses to organic contamination. This might allow for the development of specific indices of response based on the induction of these enzymes. However, a cautionary note must be added to the effect that additional information is still required on the effects of season, gametogenesis, nutrition and temperature on the components of the detoxication system.

Measurement of specific contaminant responses would complement the general biological stress responses to adverse environmental conditions such as reductions in scope for growth (Bayne et al., 1976, 1979), increases in the concentration ratio of taurine: glycine (Jeffries, 1972~ Bayne et al., 1976) and disruption of normal lysosomal functions due to stress induced lysosomal destabilisation (Bayne et al., 1976, 1979~ Moore, 1976, 1979, 1980; Moore et al., 1978}. Reductions in scope for growth and destabilisation of lysosomes have been observed in mussels experimentally exposed to WAF of crude oil as described above, as well as structural alterations in the digestive cells of the digestive gland tubules (Widdows et al., 1980b). However, these types of stress responses can be induced by a wide range of natural and anthropogenic environ- mental stressors (Bayne et al . , 1976, 1978, 1979) and are by no means limited to the deleterious effects of oil-derived aromatic hydrocarbons. Therefore, any responses which are specifically induced by organic contamination would be useful for the purposes of determining possible causation of deleterious alterations in the general indices of effect, occurring in natural populations.

Impl ica t ions of xenobio t ic me tabo l i sm for the musse l

The presence of mixed function oxygenases in bivalve molluscs implies that reactive electrophilic metabolites such as diolepoxides, in the particular case of aromatic hydro- carbons, could be produced. However, the low levels of activity for enzymic components of the detoxication system suggest that it probably does not play an important part in the removal of organic xenobiotics from the tissues. Nevertheless, diolepoxides of benzo(a)- pyrene have high mutagenic and carcinogenic activity (Levin et al., 1978} and it is considered likely that similar metabolites of other environmental hydrocarbons such as chrysenes and dibenzanthracene are also ultimate carcinogens (Jerina et al., 1978). These compounds are carcinogenic at very low concentrations in mammals (Levin et al., 1978) and this could provide an explanation for the apparent prevalence of reported haemocytic neoplasms in mussels (Farley, 1969a; Farley & Sparks, 1970~ Lowe & Moore, 1978), since the blood cells (haemocytes) would appear to have a higher potential for metabolising organic xenobiotics. However, we still require more information on the tissue distributions of polycylic aromatic hydrocarbons, as well as the tissue concentra- tions and fate of any metabolites which may be formed before we can clarify this problem of carcinogenic activity in bivalves.

CONCLUSIONS

The blue mussel, h,/. edulls, does appear to have a limited capacity to metabolise organic xenobiotics. The biological significance of this detoxication system is as yet

D e t o x i c a t i o n / t o x i c a t i o n s y s t e m s in b i v a l v e s 289

u n k n o w n a l t h o u g h t h e r e is e v i d e n c e to s u g g e s t t h a t t h e p h e n o m e n o n of e n z y m i c

i n d u c t i o n c o u l d p r o v i d e a u s a b l e i n d e x of r e s p o n s e to c o n t a m i n a t i o n b y c e r t a i n o r g a n i c

x e n o b i o t i c s .

Acknowledgements. This work forms part of the experimental ecology programme of the Institute for Marine Environmental Research, a component of the Natural Environment Research Council. It was commissioned in part by the Department of the Environment (Contract No. DGR 480/288). We are gra teful to Mrs. P. E. M. Fieth, Mrs. S. V. Mann and Mrs. S. L. Moore for skilled technical assistance and to Dr. J. F. Payne for allowing us to quote his unpubl ished data. North Sea crude oil was kindly supplied to us by Shell (U.K.) Ltd.

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