The effects of aroclor 1254 and petrochemical pollutants on cytochrome P450 from the digestive gland microsomes of four species of mediterranean molluscs

0306~4492/92$5.00 + 0.00

Camp. Biochem. Physiol.Vol. 103C. No. 3, pp. 607414, 1992

0 1992PergamonPressLtd

Printedin Great Britain

THE EFFECTS OF AROCLOR 1254 AND PETROCHEMICAL POLLUTANTS ON CYTOCHROME P450 FROM THE DIGESTIVE GLAND MICROSOMES OF FOUR SPECIES OF MEDITERRANEAN MOLLUSCS AMINADAVYAWETZ,* RAMI MANELISand LEVFISHEL~ON Institute for Nature Conservation Research, George S. Wise Faculty for Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, (Tel.: 972-3-640-9813; Fax: 972-3-642-5518) (Received 24 February 1992; accepted for publication 25 March 1992) Abstract-l. Clear and significant increase in cytochrome P450 content, was recorded for the Mediterranean bivalve Donax trunculus and the gastropod Avicularia gibbosula after accidental pollution of their habitat by oil spill. 2. The significant increase in cytochrome P450 content in Donax trunculus from polluted sites or after treatment with Aroclor 1254, was not accompanied by an increase, but rather a drastic decrease, in 7-ethoxyresorufin 0-deethylase (EROD) catalytic activity. 3. Immunoblotting, using monoclonal antibody (l-12-3) against scup cytochrome P450E, failed to reveal the existence of a hemoprotein of the P450IAl gene family, in Donax trunculus or Patella caerulea collected from polluted sites or treated with Aroclor 1254.

oxygenase components were unchanged while BPH activity was increased in the chiton Cryptochiton steNeri after exposure to fl-naphtoflavone (BNF) (Schlenk and Buhler, 1988). Similarly no change in MFO compounds but increase in BPH activity was recorded in the oyster Crassostrea oirginica (Anderson, 1978) after exposure to 3-methylcholanthrene (3MC), benz[a]pyrene, or Aroclors, and in Mytilus edulis after exposure to diesel oil (Livingstone, 1987). Increases in microsomal cytochrome P450 specific content and NADPH-cytochrome c reductase activity have been consistently observed with exposure of mytilids to polynuclear aromatic and other hydrocarbons (Gilewicz et al., 1984), and in some situations the increase was progressively greater over a range of dosage of hydrocarbons (Livingstone, 1987, 1988). Increases in microsomal NADPH-cytochrome c reductase activity have been particularly marked in the gastropods (Livingstone, 1988). Environmental pollutants, from coal, crude oil and petrochemical industry, including: polynuclear aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), chlorinated dibenzofurans, and chlorinated dibenzodioxins, usually exert their impact on biological systems by induction, of a specific P450 enzyme belonging to the gene family P450IA (Nebert and Gonzalez, 1987; Gonzalez, 1989), although post-transcriptional effects of such xenobiotics had also been reported (Goldman and Yawetz, 1990, 1991, 1992). The most studied catalytic activities associated with the induction of cytochrome 7-ethoxyresorufin 0-deethylase P450IA 1, are (EROD), and aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH). AHH activity, which is usually associated with P450IAl could be induced in many organisms, ranging from fungi to insect, including

INTRODUCTION In molluscs, cytochrome P450 and associated mixedfunction-oxidase (MFO) components are primarily present in the digestive gland, the hepatopancreas. This part of the digestive system is involved in the intracellular digestion and sequestration of heavy metals and organic xenobiotics (Moore, 1985). The high levels of lipids in this tissue make it a major site of uptake of organic xenobiotics (Widdows et al., 1983), and Lipophilic organic compounds are readily bioaccumulated by molluscs (Geyer et al., 1982). Various monooxydative activities have been reported in molluscs, including benzo[a]pyrene hydroxylase (BPH), and 7-ethoxyresorufin 0-deethylase (EROD) in Mytilus edulis (Stegeman, 1985). A number of xenobiotic exposure studies have been reported both to single compounds, and to pollutant mixtures such as crude oil. Field comparisons of populations from clean and polluted environments have also been carried out. Results have varied from no change to increase in the activity or content of various components of the cytochromes P450 system. Increase in cytochrome 450 content has not always been accompanied by a corresponding increase in enzymatic activity. Thus, despite the elevation in various components of the digestive gland monooxygenase components, no increase in BPH activity was recorded (Livingstone, 1988). In contrast, measured mono*To whom correspondence should be addressed. Abbreviations: BPH, benzo[a]pyrene hydroxylase; EROD, 7-ethoxyresorufin 0-deethlase; BNF, naphtoflavone; 3MC, 3-methylcholanthrene; PAH, polynuclear aromatic hydrocarbons; PCB, polychlorinated biphenyls; AHH, aryl hydrocarbon hydroxylase; ECOD, 7-ethoxycoumarin 0-deethylase. 607

AMINADAV YAWETZ er al.

608

reptiles amphibians and birds (Nebert et al., 1989). But there is no convincing evidence that hydrocarbons can induce cytochrome P450IAl in molluscs (Nebert et al., 1989), although chitons contain a protein immunodetected by anti-P450IAl antibody (Schlenk and Buhler, 1989). Cytochrome P450 has been partially purified from digestive gland microsomes of Mytilus edulis (Kirchin et al., 1987). The major cytochrome fraction purified from the digestive gland microsomes of Mytilus edulis had absolute oxidative absorption spectrum indicating that the P450 was largely in the low-spin state (Kirchin, 1988). Using hybridization of total RNA from digestive gland of M. edulis to rat liver cDNA probes, transcript sequences showing homology to rat P45OIVl were detected (Livingstone et al., 1989). P450IVAl is involved in the endogenous metabolism of fatty acids in rat liver (Hawkins et al., 1987) and is thought to be an ancient cytochrome P450, originating some 800 million or more years ago (Nebert and Gonzalez, 1987; Nelson and Strobel, 1987). The possibility for the existence of more than one cytochrome P450 in molluscs cannot be ruled out, two P450 fractions were resolved from M. edulis on ion exchange column, and NADPH-independent 7ethoxycoumarin 0-deethylase (ECOD) activity of Mytilus edufis revealed biphasic kinetics (Kirchin et al., 1988). Multiphasic kinetics for ECOD activity was indicative for the existence of several P450 enzymes in mammalian microsomes (Astrom and DePierre, 1986). Attempts have been made to use the content and activity of the molluscs monooxygenase system for monitoring pollutants in the environment. No difference

in

P450

content

or

BPH

activity

was

seen in Mytilus edulis from clean and polluted sites around Cape Code, U.S.A. (Stegeman, 1985). On another survey. cytochrome P450 was elevated in the same species, collected from some of the more contaminated sites along a pollution gradient in Langesundfjord, Norway (Livingstone, 1988). The present paper endeavored to characterize the MFO system in various molluscs species along the Israel coast of the Mediterranean Sea trying to identify changes in the monooxygenase system that are related to environmental pollution with polynuclear aromatic-, chlorinated-, and other hydrocarbons. The Israel Mediterranean coastline is approximately 200 km long. Two thirds of the country’s population lives within 25 km of the coastline, and 65 km of the coastline are closed areas such as ports, power plants, petrochemical industry and other industrial sources of pollutants. Part of the rivers in Israel do carry, to the Mediterranean coast, sludge that very often includes industrial waste. Dumping of industrial sludge, of extreme hazardous nature, is still done in Haifa bay where the industrial complex includes: refineries, petrochemical industry, plastic, colors, and fertilizer factories, and a major port. PCB was found to be a major pollutant in Israel (Yawetz et al., 1978, from

1989).

1983) Haifa

and bay

PCB residues are especially

in tissues of wildlife high (Dotan et al.,

MATERIALS AND METHODS

Chemicals Aroclor 1254 was purchased from Analabs, North Haven, CT. NADPH, dithiothreitol (DTT), bovine serum albumin, 7-ethoxyresorufin, nitroblue tetrazolium (NBT), 5-bromo-4chloro-3-indolylphosphate (BCIP), were purchased from Sigma. Monoclonal antibody (Mab 1-12-3) against scup P450E was a gift from J. J. Stegeman, WHOI, Woods Hole, MA, U.S.A. The secondary antibody (alkaline-phosphatase conjugated goat anti-mouse IgG) for Western blotting was from Bio-Rad. All other chemicals were of the highest commercial grade available.

Collection sites Molluscs were collected from predetermined sampling sites. Some of the collection sites as Maagan Michael were expected to be relatively clean, and served as reference to the collection sites that were set in polluted areas such as Haifa bay or Ashdod. The molluscs were brought alive to the laboratory and killed within 24 hr for determination of cytochrome P450 content and activity. Molluscs for dosing experiments were kept in the laboratory in well aerated aquaria.

Dosing procedure The dose-response studies were carried out by exposing the molluscs to the polychlorinated biphenyls mixture. Aroclor 1254. Aroclor 1254, dissolved in dimethylsulfoxide (DMSO), was introduced to the aquaria containing the molluscs in 0.2 ul DMSO uer 2 1 of sea water. Each edition of Aroclor 1254 was done on consecutive days and added 10 ppm to the final Aroclor 1254 concentration.

Preparaiion of microsomes The molluscs soft body parts were removed from the shells, the digestive gland dissected and homogenized in 5-10 volumes of 0.15 M KC1 in 50mM Tris (tris[hydroxymethyl]-aminomethane) buffer, pH 7.4, and microsomal fractions were prepared as previously described (Yawetz ef al., 1983, 1991). The microsomes were suspended in 50mM Tris buffer, pH 7.4 containing 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM DTT, and 20% glycerol, at a ratio of 0.5 ml buffer per 2.5 g of tissue wet weight. P450 content and activity determinations were done immediately or after being stored overnight at -25’C.

Enzymatic assays The content of cytochrome P450 was determined from the carbon monoxide-bound, reduced versus reduced microsomes as previously described (Yawetz et al., 1978a). assuming molar extinction coefficient of 91 mM-’ cm-’ (Omura and Sato, 1964). Measurements were made on a Jasco Uvidec 610-double beam spectrophotometer. Cytochrome b, was determined from the difference in absorbance between 424 and 409 nm in the NADH-reduced versus oxidized difference spectrum assuming an extinction coefficient of 163 cm-’ mM_’ (Garfinkel, 1958). The enzymatic activity of NADPH-cytochrome c reductase was determined by the rate of increase in absorbance at 550 nm following the reduction of cytochrome c. The reaction mixture consisted of: 0.05 M phosphate microsomal suspension containing buffer, pH 7.4, 50-100 peg of protein; 50 PM oxidized cytochrome c as an electron acceptor; and 1 mM KCN. The reaction was started by the addition of NADPH (0.1 mM final concentration) to the sample cell and the activity was recorded. Cytochrome c (reduced minus oxidized) was estimated from the extinction coefficient of 19.6 cm-’ mM-’ (Yonetani, 1965). The catalytic activity of 7-ethoxyresorufin o-deethylase (EROD) activity was measured by the spectrophotometric method of Klotz e/ a/. (1984) using molar extinction coefficient of 73 mM-’ cm-’ for the product resorufin. The

EtTects of hydrocarbon Table

on P450 in Mediterranean

609

molluscs

of the microsomal electron transport system in: POW//~caeruleo, Aviculariagibbosula, Donax lrunculw and Brachidontesvariabilis

I. Components

P-450 (pmol/mg)

Species Patella Avicularia DOnaX Brachidontes Results

pollutants

98 f 52 109 + 28 61 +46 9+8

NADPH-cytochrome c reductase (nmol/min/mg)

b, (pmollmg) 112+33 147 * 47 132+30 33+ II

13.4 9.3 IO.5 I.7

i f * +

3.2 2.8 3.1 0.9

EROD pmol/min/mg 53 24 35 48

+ + f +

25 7 39 22

are means f S.D. of 3 to I5 determinations.

reaction mixture was composed of 0.1 M Tris buffer, pH 8.0, containing 0.1 M NaCl, 2 PM substrate and lOO_2OOpg of microsomal protein in a final volume of 0.5 ml. The reaction was initiated by the addition of 0.5 mM NADPH, and run at 25°C. The formation of the product resorufin was monitored spectrophotometrically at 572 nm for 10 min. Immunoblot analysis Monoclonal antibodies (Mab l-12-3) against cytochrome P450IAl (scup P450E) were obtained by conventional hybridoma techniques and their preparation and characterization have been described (Park er al., 1986; Kloepper-Sams Ed al., 1987). Immunoblotting, as originally described by Towbin et al. (1979), was carried out essentially by the method of Burnette, 1981 with some modifications as previously described (Yawetz et al., 1991). Microsomes were separated on 613% gradient SDS-PAGE gels and transferred overnight onto 0.22pm nitrocellulose sheets at 4°C. Incubations of nitrocellulose was carried out in the presence of 5% (w/v) dry milk in Tris-buffered saline (TBS). to block nonspecific reaction. The primary antibody (Mab 1-12-3) was diluted in TBS/milk to 100 pg/ml and incubated with the nitrocellulose for 2 hr. The secondary antibody, alkaline-phosphatase conjugated goat anti-mouse IgG, was then incubated with the nitrocellulose for 1 hr. Colour was developed by incubating blots with 0.1 M NaHC02, 1 mM MgC12, buffer, pH 9.8 containing 0.33 mg/ml nitroblue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-3-indoyl phosphate, added in dimethylformamide. Development of colour was stopped within 30 min by thorough rinsing with distilled water. Statistical analysis The results are expressed as mean + S.D. The data were analyzed by analysis of variance. Prolein Microsomal protein was measured by the Bradford (1976) procedure against bovine serum albomine as standard.

RESULTS

The content and enzymatic activity of some components of the microsomal fraction from the digestive gland of the gastropods: Patella caerulea and Avicularia gibbosula, and the bivalves: Donax trunculus and Brachidontes uariabilis are presented in Table 1. Cytochrome b, content was equal or higher than that of cytochrome 450. NADPH cytochrome c reductase activity was similar in three species and much lower in Brachidontes variabilis the microsomes for which were also characterized with low content of cytochromes P450 and bS. EROD activity was recorded for each of the molluscs species, but there was no correlation between cytochrome P450 content and the rate of EROD activity. Thus Auicularia gibbosula, although possessing the highest P450 content showed the lowest EROD activity while Bracidontes uariabilis, exhibiting the lowest content of P450, still retains relatively the same rate of EROD activity as demonstrated by the other molluscs species studied here. The yield of microsomal protein was typically 4-6mg/gr wet weight. Addition of phenylmethylsulphonyl fluoride (PMSF) to homogenization buffers had little effect on the microsomal cytochrome P450 yield or spectra. Similar results were reported for the co-difference spectra (Kirchin, 1988), or benzo[a]pyrene hydroxylase (BPH) activity (Stegeman, 1985). These results are not surprising since molluscan lysosomal acid proteases were reported to belong to the cathepsin D-type proteases so that they are inhibited by pepstatin but not by thiol or serine protease inhibitors (Okada and Aikawa, 1986). The maximal wavelength of the absorbance band of the carbon monoxide-difference spectrum of

Table 2. Characterization of the spectral and catalytic properties of Donax rrunculusdigestive gland-microsomal-cytochrome P450, from different sampling sites, and control and Aroclor 1254 treated molluscs 450”m Treatment or sampling site Akko Frutarum Kiryat-Yam Control Aroclor

max. wavelength 450 450 450 450 448

CO difference spectrum band 420 nm band intensity OD/mv 45&490 nm 0.0016 0.0046 0.0160 0.0020 0.0115

max. wavelength 418 419 419 419 419

intensity OD/mg peak height 0.004 0.005 0.018 0.008 0.016

EROD pmol/min/mg 6Ok I7 9*4 0 46 ? 28 0

I. Samples from Akko, Frutarum and Kiryat-Yam were collected on the 26 August 1991. Accidental major crude oil spill occurred at sea, “ear Kiryat-Yam on the I2 August 1991. The crude oil reached the beach and polluted it for several weeks. 2. Samples of Donax from Akko seived as control, or were treated by exposure to 30 ppm final concentration of Aroclor 1254 in sea water. Aroclar was introduced, dissolved DMSO, on day 0.2, and 4. Each addition added IO ppm to the final Aroclor concentration. The molluscs were processed 5 days after the addition of the last dose of Aroclor.

AMINADAV YAWETZ et al.

610

P450 COLLECTED m Patella

CONTENT FROM

IN MOLLUSCS

DIFFERENT

*vial_.

m

SITES

_x

0

17/ 11 /Q 1 &a&i_

-aria

Achz

Akko

Frut

K-Ym

Shik

SAM’l_ING

M-MI

HaCt

T-AV

AsM

SITES

Fig. 1. Cytochrome P450 content in microsomes of the digestive gland from the gastropods: Patella caerulea and Avicularia gibbosula; and the bivalves: Donax trunculus and Brachidontes variabilis, collected on the 17 November 1991 from different locations along the Israel Mediterranean coast. The locations were, from the north to the south: Achz-Achziv; Akko; Frut-Frutarum (petrochemical plant near Akko); K-Ym-Kiryat-Yam (where a major accidental oil spill occurred on the 12 August 1991); Shik-Shikmona; M-Ml-Maagan-Michael; Hadr-Hadera; T-Av-Tel Aviv; Ashd-Ashdod. truncufus digestive-gland microsomes, presented at Table 2, was 450 nm, and 448 nm for the Aroclor 1254 treated mollusc. The co-difference spectra was also characterized by the presence of a low-wavelength peak at 418-419nm. The same codifference spectrum was exhibited by the other three species, but the P450 absorbance maxima were always at 450 nm. Cytochrome P450 content, in microsomes from the digestive gland of several molluscs species collected on the 17 November 1991 from different locations along Israel Mediterranean coast, is presented in Fig. 1. Cytochrome P450 content in the gastropod Patek caeruiea was basically the same in specimens brought from different sampling sites. The same is true for the bivalve Brachidontes variabilis in which the very low content of cytochrome P450 was similar in individuals brought from polluted site, near the harbor of Ashdod, and those brought from the nature reservation of Achziv. On the other hand, pronounced differences in cytochrome P450 content were recorded for cytochrome P450 content from the bivalve Donax trunculus. The highest P450 content was demonstrated by specimens from Kiryat Yam (K-Ym), following major crude oil spill that occurred at sea near that sampling site on the 12 August 1991. Cytochrome P450 content in the gastropod Auicularia gibbosula was also significantly (P < 0.05) higher in samples brought from Kiryat Yam compared to its content in specimens brought on the same date from Akko sampling site, located about 10 km away. Cytochrome P450 content in Patella caerulea decreased significantly (P < 0.05) towards spawning and garnet formation (Fig. 2). The decrease in P450 content was detected as early as November in individuals from sampling site located near the outlet of Hederas coal power plant, were water temperature was higher in about 10°C compared to the surroundings. Cytochrome P450 content in Patella caerulea Donax

specimens sampled at the same date but from nearby location, where water temperature was normal for the season, was still as high as recorded in August. Seasonal changes in cytochrome content in molluscs were reported for the microsomal cytochrome P450 of the digestive gland of Mytilus edulis, where the hemoprotein content declined with the approach of spawning and the eventual release of gametes and increased during autumn with the build-up of food reserves (Livingstone, 1989). A very clear and significant change in P450 content, that could be correlated with environmental pollution with polycyclic aromatic-, non aromaticand polychlorinated hydrocarbons, were recorded in microsomes of the digestive gland of Donax trunculus collected on five different dates from three locations along Haifa bay (Fig. 3). Cytochrome P450 content PATELLA TOWARDS m

cyt.

P450

P450

AM)

GAMETES 0

b5

CONTENT

FORMATION Cyt.

b5

^D

Fig. 2. Cytochromes P450 and b, content in the microsomal fraction from the digestive gland of Pate/la caerulea collected towards spawning from Hedera. H-Hedera; H-CPPsampling site near the outlet of Hederas coal power plant cooling water system. Sea water temperature at that site was higher in 10°C compared to the surroundings.

Effects of hydrocarbon P450 m

content in Donax trunculus collected from Haifa bay -

Frutarun

24/7/90

20/5/90

611

pollutants on P450 in Mediterranean molluscs

m

MO

5/l

j/90

Kiryat

261619

1

Yam

t7/11/91

month Fig. 3. Cytochrome P450 content in microsomes of the digestive gland from Donax trunculus collected on five different dates from three locations along Haifa bay. A major crude oil spill occurred on the 12 August 1991 near Kiryat-Yam sampling site.

in Donax from Akko was relatively low and was significantly (P < 0.05) lower than that recorded for the same species near the petrochemical plant Frutarum, during all sampling period, excluding the samples taken on the 26 August 1991. The cytochrome P450 level in Akko was not significantly different from its low content in Donax truncuh collected from the unpolluted site of Maagan Michael (Fig. 1). Cytochrome P450 contents of Donax truncuh sampled from Kiryat Yam (Fig. 3) until 5 November 1990 were low, and similar to the content of the hemoprotein in Donax from Akko. Following a major crude oil spill, that occurred at sea, near Kiryat Yam sampling site, on 12 August 1991, an increase of more than five times in cytochrome P450 content was recorded in Donax, sampled two weeks after the accidental SURVIVAL

OF

DCNAX

3-m -a-

c0”hc.l

of -o-

EXPOSED

Aroclor -oFI_ loppn

TO

10

oil spill (26 August 1991) and the level of this hemoprotein in samples taken two and a half months after the event, still remained significantly higher (P < 0.05) then in Donux sampled on the same date (17 November 1991) both in Akko and Frutarum. The effects of treatment with Aroclor 1254 on Donax trunculus were investigated under controlled laboratory conditions. From the survival curves (Fig. 4) it is evident that Aroclor 1254 has a toxin effect on the Donax, the exposure to the higher dose (30 ppm), was highly lethal for the mollusc. Exposure to the higher dose of Aroclor 1254 (30 ppm), caused a significant (P < 0.05) induction of about 3 times in the specific content of cytochrome P450 of the microsomal fraction from the Donax digestive gland (Fig. 5).

and

1254 -*m-OR_ 3oppn

P450 exposue m

CoNTRa

ihductim to

m 3opPm D

Domx

trunculus

Aroclor AROCLCR

1254 ,254

1

0’ 0

3

6

TIME

9

12

15

(Days)

Fig. 4. Survival curves for Donax trunculus exposed to 10 ppm and 30 ppm final concentrations of the polychlorinated biphenyls mixture_Aroclor 1254. Aroclor 1254, dissolved in dimethylsulfoxide (DMSO), was introduced onto aquaria containing about 70 molluscs each, in 0.2 ~1 DMSO per 2 1 sea water. For the 10 ppm dosage Aroclor 1254 was added on day 0, for the 30 ppm dosage Aroclor 1254 was added on days 0, 2, and 4, each addition added 10 ppm to the final Aroclor 1254 concentration.

Fig. 5. Cytochrome P450 induction in Donax trunculus exposed to the polychlorynated biphenyls mixture_Aroclor 1254. Aroclor 1254, dissolved in dimethylsulfoxide (DMSO), was introduced to the aquaria containing the molluscs in 0.2 ~1 DMSO per 2 1 of sea water. Addition of Aroclor 1254, was done on day 0, 2, and 4 and added 10 ppm to the final Aroclor 1254 concentration. The Mollusts were processed 5 days after the addition of the last dose of Aroclor 1254.

AWINADAVYAWETZ et al.

612

There was no correlation between the apparent increase in cytochrome P450 content in Donax trunculus either from polluted locations or treated with Arocior 1254, and EROD catalytic activity (Table 2). Rather the contrary high increase in cytochrome P450 was correlated with drastic decrease of EROD activity. Immunoblotting with monoclonal antibody 1-12-3 against scup cytochrome P450, E, faiIed to reveal the existence of hemoprotein of the P450IAl gene family, in the digestive gland microsomes of Donax trunculus or Patella cuerulea from polluted sites, or in the same microsomal fraction of Donax truncu~us treated with Aroclor 1254. DISCUSSION

The values of the content and specific activity of the components of the microsomal electron transport system from the digestive gland of the molluscs species studied here are within the ranges, reported for the same parameters for other molluscs species (Stegeman, 1985; Livingstone, 1987; Livingstone and Farrar, 1984; Schlenk and Buhler, 1988). The carbon monoxide-difference spectrum of the microsomal fraction of the molluscs species studied here had maximal absorbance band at 450nm, within the wavelength range, 447-452, reported for other molluscs species (Livingstone and Farrar, 1984). The presence of short wavelength peak between 418-419 nm, displayed by the molluscs studied here, was reported for other moliuscs species, at 420 nm (Gilewicz ef al., 1984) or 424 nm (Ade et al., 1982), or between 416-418nm (Livingstone et al., 1989). In Donax trunculus treated with Aroclor 1254, the carbon monoxide-difference spectrum of the digestive gland microsomes, was characterized by 2 nm blue shift of the P450 absorption band, and an increase in the intensity of the low-wavelength absorption peak (Table 2). Such changes in the carbon monoxidedifference spectrum, including 2-3 nm blue shift of the P450 peak and an increase in the size of the low-wavelength absorption peak, were reported for other molluscs species (Livingstone ef al., 1989) following treatment with polynuclear aromatic-, chlorinated-, and other hydrocarbons. On the other hand, the carbon monoxide-difference spectrum of Donax trunculus collected from Kiryat Yam, two weeks after the accidental oil spill (Table 2), was characterized by a pronounced increase of both the P450 peak and the low wavelength peak, but without blue shift of the P450 peak. It was speculated that the hemoprotein responsible for the low wavelength peak could be a breakdown product derived from cytochrome P450. This assumption is supported by finding that the major cytochrome fraction purified from the digestive gland microsomes of Myths edulis was devoid of the low-wavelength peak (Kirchin, 1988) typical for the molluscs carbon monoxide-difference spectrum. An increase in P450 content that was not accompanied with an increase in relevant catalytic activity, such as EROD, was demonstrated by Donax treated with Arocior i 254 or collected from polluted sites. Similar phenomenon was reported for various

mollusc species. Thus, no increase in benz[a]pyrene hydroxylase activity (BPH) was seen in digestive gland homogenates of Mytiius mercenaria following injection of the clam with Aroclor 1254 (Anderson, 1985), and only slight elevation of aryl hydrocarbon hydroxylase activity with exposure to PCB or other xenobiotics have been reported for mussels and other bivalves (Payne et al., 1983). No band, designating the existence of cytochrome P45OIAl in the digestive gland microsomes of Donax trunculus or Patella caerulea from polluted sites, or in microsomes of Donax trunculus treated with Aroclor 1254, could be detected when anti-scup cytochrome P450E monoclonal antibody was used as a probe. This result may indicate that no such P450 type of hemoprotein is present in Donax trunculus, or Patella caeruiea or alternatively that the antibody against the scup P450E do not recognize the cytochrome P450IAl from both molluscs species. The anti-scup cytochrome P45OE, monoclonal antibody 1-12-3, used in this study to detect the presence of a hemoprotein of the P450IAl gene family in the molluscs, was shown to recognize epitops, on the P45OIAl gene family products, in a variety of species. A relationship between scup P4SOE and rat P450,, both belonging to the P450IAl gene family, was clearly demonstrate using monoclonal antibodies to this protein (Klotz et al., 1986; Park et al., 1986). Monoclonal antibodies (Mab) 1-12-3 to scup P450E, and Mab C4 and C6 to rat P450,, each recognized the other P4501Al gene product. It was suggested (Stegeman and Kloepper-Sams, 1987) that all the teleosts BNF-(or PAH)-inducible forms represented by scup P450E, cod P450,, and trout P450LM4b are orthologous to the mammalian representatives of P450IA1, i.e. rat P450,, mouse P,-450, etc. Still the possibility that the monoclonal antibody used here, although being able to recognize epitops on many members of the P450IAl gene family, is unable to recognize the molluscs hemoprotein. Indications that such protein exists in moiluscs can be found in the literature. An indirect indication for the existence of P450IAl gene product in molluscs is that in Mytifus eduiis digestive gland microsomes

~-naphto~avone inhibited BPH activity (Stegeman. 1985). Inhibition of BPH activity by p-naphthoflavone was used in vertebrates as an indicator for the presence of the PAH-inducible cytochrome P450 (Wiebel et al. 1971). A more direct indication was presented by Schlenk and Buhler (1989) describing the presence of a protein immunodetectable by anti-P450IAl antibody in the Chiton Cryptochiton stelleri. If P450IA1 does exist in the molluscs studied here, the apparent lack of increase in EROD activity in Aroclor 1254 treated organisms or in specimens brought from polluted locations, may be due to inhibition of EROD activity by high accumulation of hydrocarbons in the microsomal fraction. Acknowledgements-Thanks to John J. Stegeman and B. Woodin from Woods Hole Oceanographic Institution, Woods Hole, MA, U.S.A. for the monoclonal antibody t-12-3 against scup cytochrome P450E and the help in the immunoblot assay. The work was partially supported by research grant from MED-POL.

Effects of hydrocarbon

pollutants

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