Liver microsomal mono-oxygenase induction in winter flounder (Pseudopleuronectes americanus) from a gradient of sediment PAH concentrations at Sydney Harbour, Nova Scotia

Marine Environmental Research 37 (1994) 283-296

Liver Microsomal M o n o - O x y g e n a s e Induction in Winter Flounder (Pseudopleuronectes americanus) from a Gradient of Sediment P A H Concentrations at Sydney Harbour, Nova Scotia R. F. Addison,* D. E. Willis & M. E. Zinck Department of Fisheries and Oceans, Physical and Chemical Sciences Branch, Bedford Institute of Oceanography, Dartmouth, Nova Scotia, Canada, B2Y 4A2

(Received 30 June 1992; revised version received 16 November 1992; accepted 18 November 1992)

ABSTRA C T Winter flounder (Pseudopleuronectes americanus) were sampled in summer 1989 and 1990 in Sydney Harbour, Nova Scotia, along a gradient of sediment polynuclear aromatic hydrocarbon (PAH) concentrations arising from industrial contamination; an uncontaminated bay nearby was used as a reference site. Hepatic mono-oxygenase activity, estimated by measurements of catalytic activity and by immunochemical analysis of cytochrome P-450 1A, increased with sediment P A H concentrations; the extent of induction between the reference and the most contaminated sites ranged up to 50-fold. Cyanoethoxycoumarin O-de-ethylase activity was at least as sensitive an indicator of mono-oxygenase induction as ethoxyresorufin O-de-ethylase or benzo(a)pyrene hydroxylase, and all three enzyme activities were well correlated with cytochrome P-450 1A concentrations and with sediment P A H concentrations.

INTRODUCTION The sediments o f Sydney H a r b o u r , N o v a Scotia (Fig. 1), are contaminated by polynuclear aromatic hydrocarbons (PAH) released over the *Present address: Department of Fisheries and Oceans, Ocean Chemistry Division, Institute of Ocean Sciences, P.O. Box 6000, 9860 West Saanich Rd, Sidney B.C., Canada V8L 4B2. 283 Marine Environ. Res. 0141-1136/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain

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Fig. 1. Map of Sydney Harbour, Nova Scotia, showing total PAH concentrations (/zg gl dry wt) in surface sediments (redrawn from Vandermeulen (1989)), and identifying cruise tracks along which fish were sampled in 1989 and 1990. The reference site, Georges Bay, is also shown. last 90 years or so from coke ovens associated with a local steel mill (Vandermeulen, 1989). A clear spatial gradient o f P A H concentrations in surface sediments exists in the harbour, from over 100 p,g g-1 total P A H close to the steel mill to less than 1 /~g g-1 in the outer harbour. The

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PAH present range from three-ringed compounds (e.g. phenanthrene) to six-ringed compounds (e.g. benzo(g,h,0perylene. Heavy metals and heterocyclic organic compounds, both probably derived from local industrial sources, are also present. In spite of contamination, the harbour supports a reasonably varied marine fauna. Recreational and some commercial fisheries exist, although some areas are now closed to fishing because of concern about the health aspects of PAH contamination. Fish hepatic mono-oxygenase induction is being used increasingly to demonstrate the sub-lethal effects of contamination by PAH and related chemicals (e.g. Addison & Payne, 1986; Stegeman et al., 1988; Van Veld et al., 1990; Sulaiman et al., 1991). At present, the most widely-used indicator of induction is mono-oxygenase catalytic activity, usually that of ethoxyresorufin O-de-ethylase (EROD) or benzo(a)pyrene hydroxylase (B(a)PH). However, other measurements of induction, including those of cytochrome P-450 1A, which catalyses EROD, and of the mRNA which codes for P-450 1A are being developed (Stegeman et al., 1988; Haasch et al., 1989; Goks~yr et al., in press; Renton & Addison, in press). In this paper, we describe mono-oxygenase activity in winter flounder (Pseudopleuronectes americanus) from sites along the Sydney Harbour PAH gradient, and discuss the relative sensitivity of various indices of mono-oxygenase induction.

MATERIALS AND METHODS The distribution of sediment PAH contamination of Sydney Harbour is shown in Fig. 1 (redrawn from Vandermeulen (1989)). This is based on 'grab' sediment samples taken in a survey of Sydney Harbour in 1981, and analysed by high-performance liquid chromatography (HPLC) with UV absorption and fluorescence detection after extraction with ethanolKOH (Matheson et al., 1983). PAH concentrations in sediments at sampling points closest to the fish sampling sites recalculated from Matheson et al. (1983) were as follows:

Zone

Outer H a r b o u r Northwest A n n Southeast Bar South A r m

Mean P A H concentration (Izg g-1 dry wt sediment) (mean +_sd, no. of samples) 0-61 + 0.34 5.45 + 2.21 10.3 42.5 + 9.3

(n (n (n (n

= = = =

3) 6) 1) 4)

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Winter flounder were sampled in August 1989 and again in September 1990 in each of these zones (Fig. 1), using a 15-m inshore research vessel with either a 3 m wide beam trawl or a 12 m long otter trawl. Georges Bay (Fig. 1), which has no obvious source of industrial contamination, was used as a reference site. Fish were caught at depths of usually less than 10 m and the trawls were made for only 10-15 min. Winter flounder usually weighing from 200 to 400 g were held in flowing sea water in tanks aboard the ship for less than 2 h before being analysed. Mono-oxygenase activity was measured in 10 000 g (post- mitochondrial) supernatants (PMS) of liver homogenates, using methods described by Addison & Payne (1986). Samples were analysed for EROD activity Burke & Mayer, 1974), 3-cyano-7- ethoxycoumarin O-de-ethylase activity CN-ECOD White, 1988) and B(a)PH (Nebert & Gelboin (1968) using 3hydroxy-benzo(a)pyrene as product). These analyses were made aboard ship, usually within 3 h of catching the fish. Proteins were analysed by the method of Lowry et al. (1951), using bovine serum albumin (BSA) as standard. Glycerol was added to PMS samples to a final concentration of 30% v/v (Tamburini et al., 1984), and samples were frozen in liquid nitrogen for later analysis. Cytochromes b5 and P-450 were analysed in microsomes prepared from frozen PMS samples as described by Edwards et al. (1988). Cytochrome P-450 1A in microsomes prepared from samples collected in 1990 was measured substantially as described by Kloepper-Sams et al. (1987); it was isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) blotted to nitrocellulose sheets and detected with a monoclonal antibody to scup (Stenotomus chrysops) P-450E (MAb 1-12-3; Park et al. (1986)), which had previously been shown to cross-react with winter flounder P-450 1A (Stegeman et al., 1987). The secondary antibody was goat anti-mouse IgG conjugated to horseradish peroxidase; this was determined by densitometry using 4-chloro-1naphthol for colour development.

RESULTS Winter flounder were obtained in adequate numbers during both surveys, except for the South Arm site in 1990, where only one fish was caught. Most fish contained maturing gonads, and would probably have spawned the following spring. None of the fish showed any obvious external effects of contamination, such as the lesions described by Stegeman et al. (1988), except for some fish from the South Arm which had small red sores on the ventral side.

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TABLE 1 Indices o f Hepatic M o n o - O x y g e n a s e Activity in W i n t e r F l o u d e r (Pseudopleuronectes americanus) Samples from Sydney H a r b o u r , N o v a Scotia, in 1989

Site (sex offish)

Georges Bay (7M, 3F) Outer Harbour (8M, 2F) Northwest Arm (3M, 7F) Southeast Bar (6M, 4F) South Arm (4M, 6F)

Fish wt (g)

LS1 (%)

PMS protein (rag g l liver)

EROD (pmol rng-1 protein rnin I)

347a _+97-7 331a ±58.2 312a ±62.9 289a +_53-0 121b ±17.2

1.34a _+0.20 !.44a _+0.25 1-80b _+0.33 1.79b _+0.31 1-46a _+0-17

71-9a ±24.0 77.8ab +27-4 76.5ab +_22.5 89.9b ±11.9 108c ±24-3

233a +_107 234a ±68.3 185a ±89-7 297a ±114 504b +130

CN-ECOD B(a)PH (prnol mg 1 (pmol rng-J protein rain 1) protein rain 1) 15.9a _+9.7 2lib +74-8 269b ±131 596c ±232 822d ±233

NA 460a +_232 889b ±413 673ab ±282 1827c ±683

Data are expressed as mean ± SD of 10 fish from each site. Data in the same column followed by the same letter do not differ significantly by t-test (P > 0.05). Enzyme activities are expressed in terms of PMS protein. LSI, liver % body wt; EROD, ethoxyresorufin O-de-ethylase; CN-ECOD, cyanoethoxy coumarin O-de-ethylase; B(a)PH, benzo(a)pyrene hydroxylase; NA, not analysed.

Table 1 shows indices of hepatic mono-oxygenase activity in 1989 PMS samples. At each site, there were only occasional differences in indices of hepatic mixed function oxidase (MFO) activity significant by t-test between sexes, so data for both sexes have been combined. Fish from the South Arm were significantly lighter than those from other sites, and had higher liver PMS protein contents. Liver as a percentage of total body weight was slightly higher in the Northwest Arm and Southeast Bar samples that at the reference site. E R O D activity in fish from the South Arm was about twice that in samples from other sites. CNECOD showed a greater range of response: activity at the South Arm was induced 52-fold over levels in fish from Georges Bay, and up to four-fold over levels in fish from the other Sydney Harbour sites. B(a)PH not analysed in PMS samples from & Georges Bay) increased about four-fold between the Outer Harbour and South Arm samples. Protein contents of microsomes prepared from frozen PMS were slightly lower in the Southeast Bar and South Arm samples than in the others (Table 2). Microsomal B(a)PH activities increased about five-fold between fish from Georges Bay and the South Arm, and cytochromes b 5 and P-450 increased about three-fold. Mean mono-oxygenase activities (EROD, CN-ECOD and B(a)PH) in the Sydney Harbour fish were well correlated with the mean sediment PAH concentrations summarised in the Materials and Methods section

TABLE 2

Indices of Hepatic Mono-Oxygenase Activity in Winter Flouder (Pseudopleuronectes americanus) Samples from Sydney Harbour, Nova Scotia in 1989

Site (Sex offish)

Georges Bay (7M, 3F) Outer Harbour (8M, 2F) Northwest Arm (3M, 7F) Southeast Bar (6M, 4F) South Arm (4M, 6F)

Microsomal protein (rag g-l liver)

Cyt. b 5 (nmol (rng I protein)

Cyt. P-450 (nmol (rag-t protein)

B(a) PH (pmol (mg-l protein min-j )

7.41a +_2.75 7.28a +_2.71 4-76b +1.49 5.67ab +1.63 5.74ab +1.45

0-050a _+0.025 0-076a _+0.053 0.071a _+0-022 0.094a _+0.068 0.164b _+0.078

0.105a _+0.063 0-170b _+0.057 0.15lab _+0.068 0-224c _+0.051 0.242c _+0.095

744a +_234 1784b +722 1767b +1112 1744b +554 3387c +966

Data are expressed as mean + SD of 10 fish from each site. Data in the same column followed by the same letter do not differ significantly by t-test (P > 0.05). Enzyme activities and cytochrome concentrations are expressed in terms of microsomal protein. B(a)PH, benzo(a)pyrene hydroxylase.

TABLE 3

Regression Coefficients (_+SE), Correlation Coefficients and P Values for Regressions of Hepatic Mono-Oxygenase Activities (Expressed in Terms of PMS Protein) in Sydney Harbour Winter Flounder (Pseudopleuronectes americanus) Sampled in 1989 and 1990 on Mean Sediment Hydrocarbon Concentrations as Described in Text

Mono-oxygenase activity 1989 analyses EROD CN-ECOD B(a)PH

1990 analyses EROD CN-ECOD B(a)PH

Regression coefficient

Correlation coefficient

P

7-13E-3 +1-41E-3 1.37E-2 _+0.46E-2 3.07E-2 !-0.58E-2

0.96

0.04

0.90

0.10

0.97

0-03

2.52E-3 i-0.47E-3 6.20E-4 i,0.39E--4 1.21E-2 !-0.26E-2

0.97

0.03

1-00

0.00

0-96

0-04

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289

TABLE 4

Indices of Hepatic Mono-Oxygenase Activity in Winter Flounder (Pseudopleuronectes americanus) Samples from Sydney Harbour in 1990

Site

n

Fish wt (g)

LS! (%)

P M S protein EROD CN-ECOD B(a)PH (mg g-l (pmol mg 1 (pmol mg -1 (pmol mg 1 liver) protein protein protein rain-1) min 1) rain-l)

Georges Bay

16 (8M, 8F)

274a l.lla 280a + 4 3 . 1 -+0.30 +123

33.4a +20.9

4.77a +3.12

66.7a +55.1

Outer Harbour

16 (8M, 8F)

389b 1.26ab _+58.4 _+0.27

272a +56.3

56.8ab +43.4

6.01a -+5.09

44.4a -+23.1

Northwest 16 Arm (8M, 8F)

340c +50.0

1.47b -+0.30

254a +48.5

55.8b +21.4

6.90a -+3.36

64.9b +18.3

Southeast Bar

349c 1.48b -+41.2 _+0.49

191b +_.26.4

98.2c -+59.6

13.9b +9.72

337c +172

208a

286a

160d

31.3c

559c

South Arm

16 (8M, 8F) 1 (M)

0.87a

Data are expressed as mean + SD. Data in the same column followed by the same letter do not differ significantly by t-test (P > 0-05). Enzyme activities are expressed in terms of PMS protein. LSI, liver % body wt; EROD ethoxyresorufin O-de-ethylase; CN-ECOD, cyanoethoxy coumarin O-de-ethylase; B(a)PH; benzo(a)pyrene hydroxylase.

(Table 3). Georges Bay fish were omitted from these correlations, as no PAH data for that site were available. Mono-oxygenase activities in 1990 PMS samples are shown in Table 4; as in 1989, there were only occasional sex differences in indices of MFO activity, so data have been grouped for both sexes. Fish weights varied slightly, with the smaller fish being found in Georges Bay and the South Arm. Liver as a percentage of body weight also varied, the heavier fish having higher values, but PMS protein was fairly constant. PMS protein. contents were appreciably higher than in 1989, for unknown reasons; the same analytical method and standards were used in both years. EROD, CN-ECOD and B(a)PH activities showed a general increase from Georges Bay to the South Arm. The generally lower enzyme activities (per mg protein) in 1990 were due partly to the higher PMS protein contents: both EROD and B(a)PH activities (per gram liver fresh weight) were similar in 1989 and 1990. However, CN-ECOD activities (per gram liver) were about 10 times higher in 1989 than in 1990. Microsomes prepared from 1990 PMS samples had variable protein contents (Table 5). Both cytochromes b5 and P-450 increased approximately three-fold between Georges Bay and the South Arm.

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R. F. Addison, D. E. Willis, M. E. Zinck

TABLE 5.

Indices of Hepatic Mono-Oxygenase Activity in Winter Flounder (Pseudopleuronectes americanus) Sampled from Sydney Harbour Nova Scotia in 1990

Site (Sex offish)

Microsomal protein (mg g-Z)

Cyt. b5 (nmol mg =l protein)

Cyt. P-450 (nmol rng-l protein)

P-450 1A (Densitometer Area Units)

Georges Bay (8M, 8F)

10.7a +4.87

0.069a _+0.016

0.182a _+0.054

0.252a _+0.098 (n=3)

Outer Harbour (8M, 8F)

9.25a +_2.60

0.134bc 5--0.037

0.277b _+0.068

0.426ab _+0.018 (n=2)

Northwest Arm (8M, 8F)

8.53ab +__2.90

0.131b _+0.013

0.304bc _+0.075

0.335a _+0.137 (n=3)

Southeast Bar (8M, 8F)

6.84b +_2.10

0.134bc _+0.048

0.347c 0.133

0.649b _+0.120 (n=3)

South Arm (1M)

13.3a

0.186c

0.615d

1.31c

Data are expressed as mean + SD. Data in the same column followed by the same letter do not differ significantly by t-test (P > 0.05). Enzyme activities and cytochrome concentrations are expressed in terms of microsomal protein. P-450 IA is expressed in densitometric area units for equal amounts of microsomal protein applied to gels.

Immunochemical analysis showed the presence of P-450 1A in the 1990 samples (Fig. 2). The amount of P-450 1A increased from Georges Bay to the South Arm (Table 5) and was well correlated with EROD, CN-ECOD and B(a)PH activities in the 10 000g samples: r = 0.91, 0-93 and 0.82, respectively; P < 0.01 for all three correlations.

DISCUSSION Hepatic mono-oxygenase activity in winter flounder showed consistent spatial changes in both the 1989 and 1990 samples. Activity increased in the sequence Georges Bay < Outer Harbour < Northwest Arm < Southeast Bar < South Arm. This trend was clearest in the catalytic activities (EROD, CN-ECOD and B(a)PH), where measurements at the 'reference' and the most contaminated sites often differed by almost 10-fold or more; however, even cytochromes P-450 and b 5 differed by up to threefold between these sites. The trend within Sydney Harbour follows quali-

Liver microsomal mono-oxygenase induction in winter flounder bO --O

t.~ bO

4~ ~ID

OO

I

I

I

I

291

I

MW STD CONTROL{

OUTER HRB {

~~

S.E.BAR{ N.WARM . { m~

ARM

SOUTH MW STD

Fig. 2. Western blot of hepatic microsomes from winter flounder (Pseudopleuronectes americanus) from various sites in Sydney Harbour, Nova Scotia, probed with anti-scup P-450E antibody MAb 1-12-3 as describedin text. Standard mixesfor molecularweight determinationwere applied to the two outer lanes; equal amounts of microsomalprotein were applied to the other lanes. tatively the total PAH content of the sediments (see Vandermeulen, 1989), and although no PAH analyses have been reported in sediments from Georges Bay, concentrations there are likely to be below 1 /xg g~ (cf. Klungs~yr et ai., 1988). It is therefore attractive to conclude that mono-oxygenase activity in the fish was induced by PAH contamination of the sediments. However, some possible confounding factors must first be eliminated. The PAH analyses with which the mono-oxygenase activities correlate were carried out in 1981, and the question arises whether that pattern of total PAH distribution would have persisted until 1989 and 1990. It probably did: PAH are among the most persistent of sediment-associated hydrocarbons (e.g. Lee & Levy, 1986) with mineralisation half-lives from microbial degradation of the order of years (Heitkamp & Cerniglia,

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292

1987). The total PAH distribution in Sydney Harbour reflects input from about 90 years' operation by the coking plant, and from related activities. It is unlikely that PAH inputs between 1981 and 1989 (which represent <10% of the cumulative PAH input) would have changed the gradient of total PAH accumulated in the sediments. There were slight differences in the composition of the PAHs at different sites in the Harbour, however, with three-ringed compounds representing a higher proportion and five-ringed compounds a lower proportion of total PAHs at the Outer Harbour and Southeast Bar sites, as summarised below (calculated from Matheson et al., 1983). Site

Outer Harbour Northwest Arm Southeast Bar South Arm

Compound groups(%) in total PAH in Sydney Harbour sediments 3-ringed

4-ringed

5-ringed

6-ringed

23 17 28 10

55 52 48 49

12 20 10 24

9 12 14 16

It seems unlikely that these qualitative differences in PAH composition would affect the capacity of the sediment PAH mixture to induce, as the total PAH concentration in sediments ranged over almost 100-fold between these sites. Second, the question arises whether metal (or other contamination) would have confounded the effects of PAH on the hepatic MFO system. Sydney Harbour sediments, especially those of the South Arm, contain elevated levels of Hg, Cd, Zn and Cu, and these are also likely to be elevated in local biota, though no data exist to confirm this (Vandermeulen, 1989). However, accumulation of these metals usually inhibits the P-450 system in animals (e.g. Clarke & Lui, 1986), and Cd, in particular, does so in fish (George, 1989). Thus, the increasing trend in mono-oxygenase activity with increasing sediment PAH concentration is not likely to be an artefact caused by the presence of metals covarying with PAH. Another potentially confounding factor in relating hepatic monooxygenase activity to sediment PAH is variation in size, sex and maturity of the fish between samples. In trout, for example, mono-oxygenase activity (illustrated by ethoxycoumarin O-de-ethylase (ECOD)) varied inversely with body size (Addison & Willis, 1982) but that relationship depended on inclusion of small fish, and the variation in ECOD activity was less than two- fold. In winter flounder from an uncontaminated bay, EROD activity also varied inversely with body weight, but only in female fish, and the relationship was weak (Edwards et al., 1988). Apart

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293

from the 1989 South Arm samples, which were about one-third the weight of the other 1989 samples, mean fish weights in either year varied less than two-fold. It seems unlikely that this could explain much of the variation in indices of MFO activity. Sex and maturity both affect hepatic mono-oxygenase activity in winter flounder (Edwards et aL, 1988; Gray et aL, 1991) but in this work, most samples contained a fairly even balance between the sexes (except for the 1990 South Arm sample). Furthermore, only a very few sporadic differences between sexes occurred in mono-oxygenase activity within any sample, as the fish were sampled at the season when reproductive maturation should have a minimal effect (see Edwards et aL, 1988). There is no obvious explanation for the year-to-year variation in mono-oxygenase activity. After allowing for the variation in PMS protein content of fresh tissue (which might have been due to deterioration of BSA standards) EROD and B(a)PH activities, calculated on a tissue fresh weight basis, were about equal in 1989 and 1990 but CN-ECOD was about an order or magnitude lower in 1990. Some of this difference might be explained by differences in maturity and some might be due to migratory movements; unfortunately, nothing is recorded about the migration of winter flounder in Sydney Harbour. Although the 1989 and 1990 samples were taken in different months, temperature differences are unlikely to explain the variation in biological response. Mean surface temperatures in the Sydney Bight outside Sydney Harbour) are around 17°C in August and around 15°C in September (Drinkwater & Trites, 1987), and the harbour waters are probably well mixed. Finally, it should be emphasised that both the 1989 and 1990 samples led to the same general conclusions about spatial trends in contamination, and that within each year's samples, measurements of mono-oxygenase activity were internally consistent. The extent of induction between the reference and the most contaminated site varied depending on the measurement used; EROD was induced two- and five-fold in 1989 and 1990, respectively, B(a)PH sixand nine-fold, and CN-ECOD 50- and six-fold. Cytochromes b5 and P-450 were induced about three-fold, and P-450 1A (estimated imunochemically) about five-fold (though this ratio was based on a single fish collected from the most contaminated site in 1990). EROD and B(a)PH are catalysed by P-450 1A (e.g. Kloepper-Sams et al., 1987) but it is not yet clear which of the P-450 isozymes catalyse(s) CN-ECOD. P-450 1A is probably involved, as CN-ECOD activity is induced by /3-naphthoflavone treatment (White, 1988; Addison et aL, 1991) but other isozymes may also participate. As this study, and some recent work (Addison et aL, 1991; Renton & Addison, in press) show that CN-ECOD induction is

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R. F. Addison, D. E. Willis, M. E. Zinck

at least as sensitive as E R O D to induction by /3-naphthoflavone and related compounds, it is obviously desirable to identify the isozyme(s) involved. In conclusion, several measurements o f hepatic microsomal m o n o oxygenase activity in winter flounder were well correlated with P A H concentrations in sediments o f Sydney Harbour. The correlation is not attributable to factors such as contaminants other than PAH, or to variation in body size or maturity o f the fish, and therefore seems likely to indicate a cause-and-effect relationship.

ACKNOWLEDGEMENTS The authors thank Dr J. J. Stegeman for a gift o f the P-450 1A probe M A b 1-12-3, and for his comments on an earlier version o f the manuscript. They also thank W. Barchard, J.M. Bewers, P.D. Keizer and J. H. Vandermeulen for reviewing the text.

REFERENCES Addison, R. F. & Payne, J. F. (1986). Assessment of hepatic mixed function oxidase induction in winter flounder (Pseudopleuronectes americanus) as a marine petroleum pollution monitoring technique, with an Appendix describing practical field measurements of MFO activity. Can. Tech. Rep. Fish. Aquat. Sci., 1505, 51 pp. Addison, R. F. & Willis, D. E. (1982). Variation of hepatic ethoxycoumarin Ode-ethylase activity with body weight and other factors in brook trout (Salvelinus fontinalis). Can. J. Fish. Aquat. Sci., 39, 924-6. Addison, R. F., Hansen, P.-D., Pluta, H.-J. & Willis, D. E. (1991). Absence of hepatic mono-oxygenase induction in estuarine fish by Ugilec-141 tR) a PCB substitute based on tetrachlorobenzyltoluenes. Mar. Environ. Res., 31, 137-44. Burke, M. D. & Mayer, R. T. (1974). Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3methylcholanthrene. Drug Metab. Disp., 2, 583-8. Clarke, I. S. & Lui, E. M. (1986). Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol., 64, 1104-10. Drinkwater, K. F. & Trites, R. W. (1987). Monthly means of temperature and salinity in the Scotian Shelf region. Can. Tech. Rep. Fish. Aquat. Sci., 1539, 101 pp. Edwards A. J., Addison, R. F., Willis, D. E. & Renton, K. W. (1988). Seasonal variation of hepatic mixed function oxidases in winter flounder (Pseudopleuronectes americanus). Mar. Environ. Res., 26, 299-309.

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