Mixed-function oxygenase activity and benzo[a]pyrene metabolism in the barnacle Balanus eburneus (Crustacea: Cirripedia)

Comp. Biochem. Physiol. Vol. 68C, pp. 55 to 61

0306-4492/81/0101-0055 $02.00/0

© Pergamon Press Lid 1981. Printed in Great Britain

MIXED-FUNCTION OXYGENASE ACTIVITY AND BENZO[a]PYRENE METABOLISM IN THE BARNACLE B A L A N U S E B U R N E U S (CRUSTACEA" CIRRIPEDIA) JOHN J. STEGEMANand HEID1 B. KAPLAN Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A. (Received 19 May 1980) Abstract--1. Microsomal cytochrome P-450-dependent mixed-function oxygenases were observed in digestive gland, intestine, cirri and gonad of the cirriped crustacean (barnacle) Balanus eburneus. 2. Levels of the microsomal electron transport components cytochrome P-450, cytochrome bs, NADPH-cytochrome c reductase and NADH-cytochrome c reductase in B. eburneus were lower than found in vertebrate liver, but like those seen in some decapod crustaceans. 3. The levels of benzo[a]pyrene hydroxylase activity were maximal at pH 7.5 and at temperatures between 15 and 30°C. Maximal activities ranged between 2 and 43 pmol 3-hydroxybenzo[a]pyrene min/mg microsomal protein as measured with a fluorescence assay. Activities were higher in summer than in winter. 4. Substrate saturation curves obtained for benzo[a]pyrene were hyperbolic, with none of the complexity seen in vertebrates. The Kms for benzol-a]pyrene metabolism in digestive gland and intestine were 6-7 x 10-7 M. 5. Compounds eluting with 3-hydroxybenzo[a]pyrene in HPLC analyses constituted almost 60% of metabolites formed by barnacle digestive gland microsomes. 9-hydroxybenzo[a]pyrene, 7,8-dihydrodiol and 9,10-dihydrodiol were formed more slowly. 6. Benzo[a]pyrene hydroxylase activity in tissue microsomes from untreated animals was inhibited more than 50% by 10 -4 M 7,8-benzoflavone. 7. Cytochromes P-450 in barnacles might be functionally related to the status of hormones regulating development and reproduction.

(Tighe-Ford & Vail, 1972; Cheung, 1974; Fyhn et al., 1977). Yet only very recently have studies (Bebbington & Morgan, 1977) demonstrated conclusively that barnacles possess the characteristic crustacean molting hormone ecdysterone (20-hydroxyecdys0ne). Functions of cytochrome P-450 in barnacles might include the metabolism of hydrocarbon pollutants in the water and also the metabolism of hormones involved in the growth, development and the unusual reproductive physiology of these animals. In the latter case barnacle MF O systems might be particularly useful for investigating how cytochrome P-450 function may be linked to reproduction in crustaceans generally, as well as those features of such linkage peculiar to barnacles. Furthermore, knowledge of cytochrome P-450 systems in barnacles may aid in understanding the susceptibility or resistance to foreign compounds that might be used to control fouling by these organisms and in devising agents that may be used for such control. In this report we present the first description of cytochrome P-450dependent M F O and other aspects of microsomal electron transport systems in cirripeds, in particular the acorn barnacle Balanus eburneus.

INTRODUCTION

Cytochrome P-450 dependent mixed-function oxygenase (MFO) reactions are involved in the biotransformation of many environmental chemicals and carcinogens and in the metabolism of endogenous compounds such as steroid hormones. Hepatic and extrahepatic cytochrome P-450 systems of aquatic vertebrates possess many properties similar to the wellstudied systems in mammals (Pohl et al., 1974; Chevion et al., 1977), but M F O systems in aquatic invertebrates have received less attention. There have been studies on a number of crustacean species (e.g. Pohl et al., 1974; Singer & Lee, 1977; James et al., 1979), although most crustaceans in which M F O systems have been studied are members of the advanced order Decapoda. There is little knowledge of these systems in other types of crustaceans, a large, diverse and complex group of animals. Barnacles (subclass Cirripedia) (Barnes, 1968) comprise a unique and important group in the class Crustacea. Exclusively marine and having world-wide distribution and great abundance, they are the only group of exclusively attached crustaceans. All ordinary barnacles (order Thoracica) possess calcareous plates and are hermaphroditic, both unusual characters among crustaceans. In addition to possessing characteristics that suggest a unique crustacean physiology, thoracican barnacles present a serious problem as the most economically important group of marine fouling organisms. Because of these features, factors regulating growth, development and reproduction in barnacles have received a great deal of attention

MATERIALS AND METHODS Chemicals

Benzo[a]pyrene (BP; Gold Label) and 7,8-bcnzoflavone (7,8-BF; ~-naphthoflavone) were obtained from the Aldrich Chemical Co., Milwaukee, W1. [3H]BP was obtained from New England Nuclear, Boston, MA. Aminopyrine (AP) was obtained from Aldrich and was twice recrystallized 55

56

JOHN J. STEGEMANand HEIDI B. KAPLAN

from ethanol. Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP), reduced NADP (NADPH), reduced nicotinamide adenine dinucleotide (NADH), HEPES buffer and horse heart cytochrome e were obtained from the Sigma Chemical Co., St Louis, MO. Sodium succinate was from Fisher Scientific, Medford, MA. Materials for HPLC were obtained as before (Tjessum & Stegeman, 1979). Animals Acorn barnacles (B. eburneus), were collected from the submerged portion of styrofoam or wood rafts at local marinas in Falmouth Harbor, Falmouth, MA and Eel Pond, Woods Hole, MA, between September 1977 and December 1979. Some barnacles were maintained in aerated water from the collection site until sacrifice within 3 hr; others were held in 251 tanks with running, aerated seawater, ambient temperature 4°C, for up to three weeks before use. The soft-body parts of the acorn barnacles were about 1 cm in diameter and weighed about 0.2 g. In late fall and winter the digestive gland samples also contained small amounts of muscle and testicular tissue. Identity of tissues used was determined by histological assessment of samples fixed in formalin. There was no experimental treatment of these animals. Enzyme preparation Animals were removed from their calcareous shells and dissected tissues were placed in ice-cold 0.065 M phosphate buffer, pH 7.0. Fluids were rinsed from intestine. Tissues were minced and homogenized as previously described (Stegeman et al., 1979) in five vols of 0.065 M potassium phosphate buffer, pH 7.0, containing 1.15% KCI and 3 mM MgC12. Subcellular fractions were prepared by centrifuging at 750 g x l0 min (nuclear), 10,000 g × 10 min (mitochondrial) and 40,000 g × 90 min (microsomal). Pellets were washed and supernatants combined at each step. Nuclear and mitochondrial pellets were resuspended in 2.0 ml and microsomal pellets in 1.0 ml of 0.1 M phosphate buffer (pH 7.3)/g of tissue. Enzyme assays NADPH-cytochrome e reductase was assayed according to Phillips & Langdon (1962), using a reaction mixture containing 0.175 mM NADPH, 80/~M horse heart cytochrome c, 0.2 M phosphate buffer pH 7.7, ionic strength 0.58 in a total volume of 1.65 ml, both with and without l mM KCN. Reaction conditions at 25C and reduced cytochrome c determination were as before (Stegeman et ai., 1979). NADH-cytochrome c reductase was assayed in a similar fashion with 0.25 mM NADH replacing NADPH. Reference cuvettes in both cases contained reaction mixtures with no enzyme. Succinate cytochrome c reductase (EC 1.3.99.1) was assayed according to Green et al. (1955). Reaction mixtures, 1.65ml volume, contained 10mg/ml bovine serum albumin, 80/IM cytochrome c and 1 mM KCN in 0.02 M phosphate buffer pH 7.4. Reactions were initiated by addition of sodium succinate to 5 mM. BP hydroxylase was assayed in a 0.5 ml reaction mixture containing 0.15-0.28 mg microsomal protein and an artificial NADPH-generating system or 1.0 mg/ml NADPH or NADH, in 0.1 M Tris-HCl, final pH either 7.6 or varied as required. Reactions were initiated by addition of BP in 20~d MeOH to a final concentration of 60pM, with or without 7,8-BF. Incubations were for 30 min at either 30°C or varied temperature; activity was quite linear up to 60 min and 600/lg protein per ml. Fluorometric assay of metabolites was carried out according to the method of Nebert & Gelboin (1968) as modified by Stegeman et al. (1979). "Fluorescence" inherent in the assay, i.e. that observed when buffer alone was treated as a sample, was subtracted from each value. AP demethylase was assayed by measuring formaldehyde generation using a reaction

mixture described before (Stegeman & Binder, 1979). Incubation was for 15 min at 30°C. Reaction blanks for both assays consisted of reaction mixtures without NADPH. Cytochrome P-450 was assayed by Na2S204-difference spectra of CO-treated samples as previously described (Stegeman & Binder, 1979), in resuspensions containing about l mg microsomal protein per ml. Cytochrome b5 was estimated in similar dilutions using either Na2S204, NADPH or NADH as reductant as previously described (Stegeman & Binder, 1979). Practical limits of detection for the assays were: cytochrome P-450, 50pmol/ml of microsomal resuspension; cytochrome bs, 20pmol/ml of microsomal resuspension; cytochrome c reductases, 0.01 nmol cytochrome c reduced/ min; AP demethylase, 0.5nmol formaldehyde; BP hydroxylase, fluorescence equivalent to 1.5 pmol 3-OH-BP. Extraction efficiency for BP metabolites here and in HPLC analyses was 50~o, and the data reflect this. Protein was determined according to Lowry et al. (1951). High pressure liquid chromatography Metabolites of [3H]BP were obtained by in vitro incubation using digestive gland microsomes prepared from 160 individuals collected in winter. One milliliter reaction mixtures containing 60/~M BP, 0.22 mg NADPH and 1.4-3.5 mg microsomal protein were incubated for 20 or 30min and the metabolites were extracted and prepared according to Selkirk et al. (1976) with final dissolution in 40 or 50pl acetonitrile. Zero time and boiled enzyme blanks were treated in a similar fashion. Separation of BP metabolites was achieved using a DuPont LC 850 chromatograph fitted with a l0 ~l injection loop, a 25 cm DuPont Zorbax ODS column and filter photometer u.v. detector operating at 254 nm. Elution of metabolites was performed by running a gradient from 40-80~o acetonitrile in water. Metabolites were identified based on co-elution with authentic standards (Tjessum & Stegeman, 1979) and quantified by counting the labelled fractions eluting from the columns. All procedures were carried out under red light.

RESULTS The subcellular distribution and characteristics of barnacle M F O systems were determined using digestive gland and intestinal tissues. In both, N A D P H and N A D H - c y t o c h r o m e c reductase activities and also BP hydroxylase activity sedimented in that fraction (Fig. 1) corresponding to microsomal preparations using vertebrate (fish) liver (Stegeman et al., 1979). Succinate-cytochrome c reductase activity was principally in mitochondrial fractions and there was little mitochondrial contamination of the microsomal fractions. Native protein, 0 2 and N A D P H were required for full M F O activity in barnacles (Table 1). There was strong inhibition of BP hydroxylase activity by CO, but about 30% appeared to be CO-insensitive, the reasons for which are unknown. BP metabolism supported by N A D H was 25 35% of that supported by N A D P H , somewhat higher than seen for this activity in blue crab (James et al., 1979). The activity when both coenzymes were present was not, however, synergistic, nor even additive in the case of intestine. BP hydroxylase activity was maximal at pH 7.6 for both tissues (Fig. 2) and the activity in each tissue was reasonably constant over a broad temperature range from 15 to 30°C, probably reflecting adaptation of these animals to the lower intertidal habitat where

57

MFO activity in barnacles " 8-

SUCCINATE-CYT.

c

RECUCTASE

NADH-CYT, c

NADPH-CYT. c

REDUCTASE

REDUCTASE

BENZO[o]PYRENE HYDROXYLASE 8

2 6-

654-

<~

t

-

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50

PERCENT

50

0

TOTAL

I

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--1

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PROTEIN

Fig. 1. Subcellular distribution of cytochrome c reductase and MFO activities in Balanus eburneus tissues. Relative specific activities were calculated according to DeDuve et al. (1962). Key: (1) nuclear, (2) mitochondrial, (3) microsomal and (4) cytosolic fractions. The distributions shown for both NADPHand NADH cytochrome c reductase activities were the same whether or not KCN was included in the reaction mixture. they are subject to extreme temperature fluctuations on small and large time scales. Dithionite reduction of CO-treated microsomes from both digestive gland and intestinal tissues resulted in peaks at about 450 nm and 424 nm and a trough at 410-411 nm. Appearance of the peak near 450 nm was dependent both on reduction and on CO, indicative of cytochrome P-450. The Soret absorption maximum for this peak in digestive gland microsomes was close to 449 nm. The peak at 424 nm and trough at 411 nm appeared upon reduction of microsomes with N A D H and without CO, clearly indicating the presence of cytochrome bs in these tissues. Cytochrome bs was reduced equally well by N A D P H as by N A D H , suggesting either that NADH-cytochrome b5 reductase readily accepts electrons from N A D P H

too or that N A D P H - c y t o c h r o m e c reductase readily transfers electrons to cytochrome bs. Analogy with mammals (Enoch & Strittmatter, 1979) would support the latter pathway. Cytochromes P-450 and bs were present in the greatest content in microsomes, but spectral interference by putative cytochrome oxidase prevented quantifying them in mitochondrial fractions. The levels of microsomal electron transport components and M F O activity in selected barnacle tissues obtained in summer are given in Table 2. Along with the highest levels of cytochrome P-450 and N A D P H cytochrome c reductase activity and BP hydroxylase activity, digestive gland had the highest levels of cytochrome bs (0.02 nmol/mgT and N A D H - c y t o c h r o m e c reductase activity ( l l 2 n m o l / m i n / m g ) . The levels of

Table 1. Some requirements for microsomal benzo[a]pyrene hydroxylase in B. eburneus Conditions

Digestive gland Units/mg (N)* "~o

Complete (NADPH) 8.2 + 0.2 Minus NADPH <0.2 + 0.0 CO-bubbled 2.2 + 0.8 N2-bubbled, N2 atmosphere 2.8 Boiled microsomes <0.2 Complete (NADH) 2.0 + 0.5 Complete (NADH + NADPH) 9.2 ___0.3

(3) (2)

100 <3

(3)

27

(1) (1) (3) (3)

47 <3 26 113

Intestine Units/mg (N)*

"o

7.2 + 0.5 <0.2 + 0.0 2.4 + 0.2 3.0 _ 0.6 <0.2 1.6 6.8

100 <3 32 41 <3 34 96

(2) (2) (2) (3) (1) (1) (1)

* Units are pmol 3-OH-BP equivalents produced per min. Data represent means + range of determinations on N separate pools of tissue with 60-80 individuals per pool using late fall or winter samples.

58

JOHN J. STEGEMAN a n d HEIDI B. K A P L A N

Table 2. Microsomal enzymes in tissues of B. eburneus Microsomal yield (mg protein/g tissue)

Tissue* Digestive gland Intestine Cirri Testes, mature Testes, immature Ovary, mature Ovary, immature

4.7 3.8 4.8 3.7 2.8 3.8 5.5

Cytochrome P-450 (nmol/mg)

_ 1.4 (4):~ + 1.0 (26) + 1.6 (8) +_ 0.l (1) + 0.1 (3) -I- 0.4(1) + 1.5 (8)

0.11 0.09 0.01 0.02

NADPH-cytochrome c reductase (units/mg)¢

Benzo(a)pyrene hydroxylase (units/mg)*

28.6 + 10.0 (3) 15.6 + 4.4 (3) 4.7 __+1.8 (2) 11.6 _+ 0.9(2) 14.0 + 1.6(1) 7.0 + 1.0(1) --

43.2 + 0.8 (3) 18.0 + 0.2 (3) 8.0 + 0.1 (2) 12.8 _+ 0.1 (2) 8.6 _ 0.3(1) 6.0 + 0.4(1) 2.0 + 0.5 (1)

+ 0.01 (3) + 0.03 (4) + 0.005 (3) __+0.005(1) ND ND ND

* From barnacles sampled in summer, except for immature ovaries which were sampled in winter. t Units are nanomoles cytochrome c reduced/min or pmols of 3-OH-BP-equivalents produced per min. :~(N) indicates number of pools assayed with more than 80 individuals in each pool. Where N is greater than two values are +SD. Other values are + range in the assays. ND = Not detected. cytochrome b5 and N A D H - c y t o c h r o m e c reductase activity in intestinal microsomes were 0.01 nmol/mg and 92 nmol/min/mg, respectively. Cytochrome P-450 was detectable in mature testis, but not in immature testis collected at the same time from other individuals in the population. Although the activity in the summer animals was rather low, barnacles sampled from the same populations in the winter had lower activity of BP hydroxylase, i.e. 9, 7 and 3 units per mg microsomal protein for digestive gland, intestine and cirri, respectively. We could not detect aminopyrine demethylase activity in any of the samples (winter) we analyzed. The low M F O activity apparently was not due to endogenous inhibitor as may exist in spiny lobster hepatopancreatic microsomes (James et al., 1979). Addition of up to 1 mg of digestive gland or intestine microsomal protein to 1 ml incubations with fish hepatic microsomes (Stenotomus versicolor; Stegeman et al., 1979) produced an additive effect on BP hydroxylase activity, rather than inhibition. Substrate saturation curves seen for BP with both digestive gland and intestine microsomes were hyper-

bolic (Fig. 3), showing no evidence of the sigmoidal character seen using similar high concentrations of fish hepatic microsomes (Stegeman et al., 1979). Even though the protein concentrations in these assays were rather high, the turnover of BP was inherently low with these microsomes, which should alleviate some factors contributing to kinetic complexities with BP (Robie et al., 1976). K,, values were similar (Table 3) to values obtained with low concentrations of hepatic microsomes from fish (Stegeman et al., 1979) and mammals (Nebert & Gelboin, 1968). BP hydroxylase activity in both digestive gland and intestine microsomes from animals sampled in summer was inhibited by 7,8-BF in vitro (Table 4). Monohydroxy and dihydroxy BP metabolites formed by digestive gland microsomes and that were clearly identifiable on the basis of coelution with standards included 9,10-dihydrodiol, 7,8-dihydrodiol, 9-OH-BP and 3-OH-BP (Fig. 4). There was a very small peak of absorbance at 254 nm that eluted with 4,5-dihydrodiol, but it contained insufficient 3H for quantification. The principal metabolite formed by

20

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Fig. 2. Dependence of barnacle microsomal BP hydroxylase activity on temperature and pH. Samples used were obtained in the winter. Data are means of replicate determinations on two samples, + range (T), or replicate determinations on a single sample (pH). Reactions were incubated for 10 min. Triangles represent data obtained with Tris rather than phosphate buffers. Closed symbols, digestive gland; open symbols, intestine.

MFO activity in barnacles

59

Table 3. Kinetic parameters of benzo[a]pyrene hydroxylase in B. eburneus microsomes* Sample microsomes

Protein concentration

Molar Km

Vm,xt

430#g/ml 410#g/ml

7.3 x 10 -7 (+3.9) 6.6 x 10-7(+2.4)

8.2(+0.8) 5.4(+0.3)

Digestive gland Intestine

* Values are means of determinations on microsomes from three separate pools of tissues with 80 barnacles in each pool. Samples were obtained in winter. Numbers in parentheses are + range. t Units are pmol 3-OH-BP equivalents produced per min per mg microsomal protein.

these microsomes prepared in winter appeared to be 3-OH-BP. As seen from the rates of formation presented in Table 5 this fraction amounted to almost 609/0 of the total mono- and dihydroxy derivatives.

While there may be other metabolites eluting with 3-OH-BP (Tjessum & Stegeman, 1979), the rates of formation of products in this [3HI peak were comparable to the rates obtained using the fluorescence

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[BENZO{o}PYRENE] (p.M) Fig. 3. Substrate saturation profiles for microsomal BP hydroxylase. Tissues were from animals sampled in winter. Data represent means of three separate determinations + range. Closed circles, digestive gland; open circles, intestine. B(a)P

40

3-OH

8

½

9 -OH 6 El_ (.~

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7,8-Diol 9,10-DioI ~

X~ 6,12- Q

2 li,l,I,,, I, I,, hl,l,,,,,,,,,,n,,,,ll

o~ o

5

to

15

20

I II,ll,[,,lllHHIil,, 25

50

55

40

ELUTION TIME (MINUTES)

Fig. 4. HPLC elution of [3H]benzo[a]pyrene metabolites formed by Balanus eburneus digestive gland microsomes. Procedures are described in Materials and Methods section. Label appearing in analyses of cofactor blank reactions was subtracted. The total label eluting with unmetabolized BP in the analysis depicted was about 4.0 x 105 CPM. Less than 2.0% of the substrate was metabolized in 30 min. Abbreviations: diol; dihydrodiolbenzo[a]pyrene. Q; benzo[a]pyrene quinone. OH; hydroxybenzo[a]pyrene.

60

JOHN J. SXEG~MANand HEIDIB. KAPLAN

Table 4. Influence of 7,8-benzoflavone on B. eburneus BP hydroxylase activity 7,8-benzoflavone concentration 0 1.25 × 10-TM 5.0 x 10-6M 1.0× 10-4M

Percentage Activity Remaining: Digestive gland Intestine 100±7" 90+3 76± ll 44±8

100± II 90±2 74±8 42±1

* 100°~, activities are like those in Table 3. Values are means of replicate determinations on a single pool of 80 individuals, ± SD of the assay. Table 5. Benzo[a]pyrene metabolite formation by B. eburneus digestive gland microsomes Metabolite

pmol/min/mg microsomal* protein

9,10-dihydrodiol 7,8-dihydrodiol 9-hydroxy 3-hydroxy

0.9 ± 1.7 ± 2.3 ± 7.0 ±

0.2t 0.l 0.3 0.4

* Assays of microsomes sampled in winter. t Values are __+range of results of replicate assays carried out using aliquots of the same microsomal fraction prepared from 160 individuals. assay. 1,6-quinone, 3,6-quinone and 6,12-quinone were also observed in the HPLC analyses, but they appeared as well in zero-time blank reactions and their significance to the overall rate of metabolism has yet to be determined. There was also an unidentified peak of [3H] activity appearing between 9,10-dihydrodiol and 7,8-dihydrodiol (Fig. 4), but this [3HI peak did not have a corresponding peak of absorbance at 254 nm and may not be a metabolite. DISCUSSION More than 25 years ago Koe & Zechmeister (1952) noted that barnacles could accumulate polynuclear aromatic hydrocarbons (PAH) from the environment. The results here establish that cytochrome P-450dependent MFO, capable of metabolizing such compounds, are present in cirriped crustaceans. With recent findings in copepods (Waiters et al., 1979), the results suggest that such systems may be generally present throughout the Crustacea. The levels of cytochrome P-450 and NADPH--cytochrome c reductase activity of microsomes from barnacle tissue were in the range of values obtained with decapod species including the lobster, H o m a r u s americanus (Elmamlouk et al., 1974), spiny lobster, Panulirus argus and blue crab, Callinectes sapidus (James et al., 1979), but were lower than seen in liver of most aquatic vertebrates (Pohl et al., 1974; James et al., 1979). In vitro MFO activity in barnacle tissues was low, like that seen in other crustaceans (Khan et al., 1972; Pohl et al., 1974; Burns 1976; James et al., 1979). Rates of xenobiotic metabolism in vivo might be low enough to preclude much advantage in the face of large contamination by hydrocarbons. Even though the activity of BP hydroxylase in these untreated barnacle tissues was low, it was inhibited by

7,8-BF. Singer et al. (1980) have also observed an exceptionally strong inhibition of BP hydroxylase activity by 7,8-BF in untreated blue crab. 7,8-BF in vitro can act to discriminate between different mammalian cytochromes P-450, inhibiting those induced by PAH (Weibel et al., 1971; Atlas et al., 1976). Possibly the constitutive cytochromes P-450 in some Crustacea are naturally sensitive to 7,8-BF, but such inhibition seen in barnacles could reflect an inadvertent and widespread induction by environmental chemicals. The question of whether or not 7,8-BF inhibition of BP hydroxylase in barnacles indicates cytochromes P-450 induced by environmental chemicals is like similar questions that exist for some fish (Stegeman & Binder, 1979), questions that have yet to be resolved. The seasonal differences seen in MFO activity in barnacles could also reflect induction, possibly related to a higher input of petroleum hydrocarbons from local boat traffic in summer. However, temperature and hormonal status might be determinants of seasonal differences. Moreover, it remains to be demonstrated that these animals in fact have the capacity to respond to foreign compound inducers. The metabolites of BP produced by barnacles do not appear to be unusual and the metabolism at the 7,8- and 9,10-positions suggests that barnacles may possess the capacity to form mutagenic diolepoxides of BP. The pattern of BP metabolites produced by B. eburneus digestive gland can be compared with that which Singer et al. (1980) report as formed by stomach microsomes from the decapod C. sapidus. Phenols constituted almost 80~o of the metabolites in the barnacle and more than 95% in the crab and in each case 3-OH-BP appeared to be the dominant component. Singer et al. (1980) also observed formation of 4,5-dihydrodiol in the crab while little or none of this product was formed by barnacles. On the other hand 9,10-dihydrodiol was not detected in the blue crab but 9-OH-BP, which can result from the rearrangement of 9,10-oxide (Selkirk et al., 1976), was found. Thus the crab, as well as the barnacle, appears to have the capacity for metabolism at the 9,10-position and the lack of 9,10-dihydrodiol in the crab could stem from other metabolic differences. Whether the patterns of metabolite formation seen in either case occur consistently must be confirmed. It is possible, for instance, that microsomes prepared from barnacles in summer might produce metabolite profiles different from those seen here for winter animals. Very low rates of xenobiotic metabolism in vitro could imply functions other than oxidative xenobiotic metabolism for cytochromes P-450 in barnacles. The seasonal and maturational (in testis) differences seen here and the sex differences and correlations between molting cycles and levels of MFO activity in blue crab (Singer & Lee, 1977), argue that some crustacean cytochromes P-450 are functionally related to hormonal status. Bollenbacher et al. (1977) and Feyereisen & Durst (1978) recently observed that cytochrome P-450 in peripheral tissues of insects catalyzes hydroxylation of ecdysone to 20-hydroxyecdysone, the most active form of molting hormone in insects (Gilbert & King, 1974) and in many crustaceans (Bebbington et al., 1977). Further studies in barnacles should reveal the roles of cytochromes P-450 in ecdysteroid and possibly sex steroid metabolism in this important group.

MFO activity in barnacles But there may also be a role for cytochrome P-450 in metabolism of molt inhibiting h o r m o n e s in these and other crustaceans. For example, insect juvenile hormones, which can inhibit molting in crustaceans, contain an epoxide that might be synthesized in a cytochrome P-450-dependent reaction.

Acknowledgements This research was supported in part by National Science Foundation grants OCE76-84415 and OCE77-24517 and the Ocean Industries Program, W.H.O.I. Metabolite standards were provided by the NCI Carcinogenesis Research Program. We thank Richard E. Wolke for histological analyses and Bruce Woodin for expert assistance with HPLC analyses. Isabelle Williams confirmed the identity of B. eburneus and Robert Gagosian and Rudolf Scheltema made useful comments on the manuscript. Contribution No. 4616 from the Woods Hole Oceanographic Institution.

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