Assessment of the impact of organic pollutants on goby (Zosterisessor ophiocephalus) and mussel (Mytilus galloprovincialis) from the Venice Lagoon, Italy: Biochemical studies

Marine Environmental

Research 39 (1995) 235-240 Elsevier Science Limited Printed in Great Britain 0141-1136/95/$09.50

0141-1136(94)00055-7

Assessment of the Impact of Organic Pollutants on Goby (Zosterisessor ophiocephalus) and Mussel (Mytilus gulloprovinciulis) from the Venice Lagoon, Italy: Biochemical Studies David R. Livingstone,” Philippe Lemaire,a Anne Matthews,a Laurence D. Peters,a Cinta Porte u Patrick J. Fitzpatrick,b Lars Fiirlin,” Cristina Nasci,d Valentino Fossato,d Nicola Woottone & Peter Goldfarb’ “NERC Plymouth Marine Laboratory, Citadel Hill, Plymouth, UK, PLl 2PB bDepartment of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Republic of Ireland ‘Department of loophysiology, University of Giiteborg, S-41390 Giiteborg, Sweden ‘Istituto di Biologia de1 Mare C.N.R., Caste110 1364/A, Venice, Italy ‘School of Biological Sciences, University of Surrey, Guildford, UK, GU2 SXH

ABSTRACT The use of cytochrome P4501A (CYPlA) and other measurements as biomarkers was investigated in liver of goby (Z. ophiocephalus) and digestive gland of mussel (M. galloprovincialis) from several sites in the Venice lagoon as part of the UNESCO-MURST Venice Lagoon Ecosystem Project. Most tissue contaminants (PAHs, PCBs, DDTs) and biochemical measurements varied seasonally. Elevated 7-ethoxyresoruJn 0-deethylase activity and CYPlA-protein levels in goby were correlated with high tissue contaminant levels at the industrial Porto Marghera site. On occasions, activities of the antioxidant enzymes catalase and putative DT-diaphorase (resorufin reductase activity) in male but not female goby were also higher at Porto Marghera than other sites, but no d@erences were seen in superoxide dismutase (SOD) activity. A range of measurements (SOD, catalase, NADPH-cytochrome c reductase and glutathione S-transferase activities, P450 and ‘418-peak’ contents) in mussel showed little dtgerence between sites. However, indications were obtained of elevated levels of CYPIAl-like mRNA, CYPIA-like protein and metabolism of benzo[a]pyrene to free metabolites in mussels from the Venice lagoon compared to a site in the Adriatic Sea. The studies demonstrate the usefulness of CYPIA as a biomarker for organic pollution in fish and indicate some potential for its application in molluscs. 235

236

D. R. Livingstone et al.

The integrated use of biomarkers and chemical contaminant levels is advocated as an effective means of pollution monitoring (Stegeman ez al., 1992; Walker & Livingstone, 1992). Such an approach was applied as part of the UNESCOMURST Venice Lagoon Ecosystem Project to assess the state of pollution in the Venice lagoon. Hepatic cytochrome P4501A (CYPlA) was measured in goby (Zosterisessor ophiocephalus) as a well-established biomarker for organic pollution, particularly polynuclear aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs) (Stegeman et al., 1992; Walker & Livingstone, 1992). Hepatic antioxidant enzymes (superoxide dismutase [SOD], catalase, putative DT-diaphorase) were also measured as potential indicators of chemical-mediated oxidative stress (Stegeman et al., 1992). No equivalent to CYPlA has yet been adequately characterised in marine invertebrates and therefore a number of possible pollutant-related biochemical responses were measured in digestive gland of mussel (Mytilus galloprovincialis), including the mixed-function oxygenase system, antioxidant enzymes and glutathione S-transferase (GST) (Livingstone, 1991). Chemical body burden was assessed in terms of tissue contaminants (aliphatic hydrocarbons, PAHs, PCBs and DDTs). Animals were collected at three times (male and female goby - April, June, October) or four times (mussel - as for fish plus December) of the year from a number of putative clean and polluted sites. These included Lio Grande and Crevan (N. Venice lagoon) and Porto Marghera (central lagoon - industrial) for goby, and Platform site (Adriatic Sea), Crevan and C.V.E. (central lagoon industrial) for mussel. Following lyophilisation, hexane extraction and alumina/ silica gel chromatography, hydrocarbons, PCBs and DDTs were measured by fluorescence HPLC and gas chromatography (Fossato et al., 1989). Cytosolic (9 000 or 1OOOOOg)and microsomal fractions were prepared by standard procedures in 0.15 M KCl/ 1 mM EDTA pH 7.5 (goby) or 10 mM Tris-HCl/O.S M sucrose/O. 15 M KC1 pH 7.6 (mussel) (20% w/v glycerol included in microsomal buffer) (Livingstone, 1988; Livingstone et al., 1992). Enzyme activities or contents were measured fluorometrically: 7-ethoxyresorufin 0-deethylase (EROD - indicator of CYP4501A) (Burke & Meyer, 1974); spectrophotometrically: SOD (EC 1.15.1.1) (Livingstone et al., 1992), catalase (EC 1.11.1.6) (Livingstone et al., 1992), putative DT-diaphorase (EC 1.6.99.2) (NADH-dependent resorufin reductase) (Nims et al., 1984), total cytochrome P450 (Livingstone, 1988); 418-peak (Livingstone, 1988), NADPH-cytochrome c reductase (Livingstone, 1988), and GST (substrate: I-chloro-2,4_dinitrobenzene) (Fitzpatrick et al., 1995); and radiometrically: benzo[a]pyrene (BaP) metabolism in presence of NADPH (3H-BaP free metabolites and protein adducts by HPLC and protein precipitation) (Lemaire et al, 1993). Western blotting for CYPlA was carried out as described by Towbin and co-workers (Towbin et al., 1979) using polyclonal anti-P4501A from perch (Perca juviatilis) (alkaline phosphatase visualization, quantification by image analysis). Analysis of CYPlAl-like mRNA was carried out by RNAzol B extraction of RNA and Northern blotting using 32P-radiolabelled cDNA probe for CYPlAl (pfP1450-3’ from trout) as described in Wootton et al., 1995. Values are given as mean f SEM and were compared by oneway analysis of variance (p < 0.05). Preliminary results for goby (biochemical data for male only shown) and mussel are presented in respectively Tables 1 and 2. Seasonal ranges of measurements are given in parenthesis in the tables. Tissue contaminant levels changed seasonally in

1902 (1240-2727) 1868 (1360-2250) 11391 (7200-18110)

137 (87-197)

122 (1-217) 127 (1-195) 430 (2-827)

DDTb

14.1 f 3.4A (5-14) 3.0%0.7B (3-5) 39.6 f 0.2C (18-142)

EROD’

Catalase’

0.89 f 0.4AB (1-13.5) 0.6*0.2A (l-7) 2.5 f 0.4BC (2.510.2)

Resorufin reductased

11.2f 0.9A (5-72) 19.51t l.lB (2-30) 46.0 * 5.5C (3-51)

dismutas$

189+25A (189-340) 353rt31c (353-499) 311 l 49AC (31 l-439)

&peroxide

TABLE 1 (EROD) and Antioxidant Enzyme Activities in Liver of Male Goby (Z. ophiocephalus) from Sites in the Venice Lagoona

u Chemical contaminants: single samples taken of either sex, average value for year given plus seasonal range in parenthesis; enzyme ativities: means f SEM (n = 3 to 7) for April 1992 sample plus seasonal range of means for each sampling time given in parenthesis; data in the same Eolumn sharing the same letter (A, B, C) do not differ significantly (p > 0.05). ng -’ dry, wt. ’ pm01 mid’ rng-’ protein. d Putative DT-diaphorase activity in nmol min-’ g wet wt. ’ mm01 min-’ 9” wet wt; /SOD units g* wet wt.

(5z4) 161 (57-233)

PCBsb

0-Deethylase

PAHsb

Levels, 7-Ethoxyresorufin

Port0 Ma&era

Crevan

Lio Grade

Site

Contaminant

8 op % ‘2

(138-538) 622 (167-1185) 814 (460-1558)

339

UCMb

(15::49)

149 (71-252) 132 (6&275)

PA Hs’

(12:;?587)

185 (145-240) 189 (133-234)

PCBs’

~~_.____

658 + 27

653 f 56

634 * 45

SODd

1.8ztO.l

1.6+10.1

1.8*0.1

Catalase

65.9i

1.7

63.9 f 6.1

57.4 zt 9.6

P45d

33.0~2.8

27.0 * 1.4

27.5 zt 2.5

‘4/g-peak’*

1.79 * 0.09

1.98 * 0.09

2.13kO.07

GSF

39.5 LIZ 0.9*

32.7 f 12.8

10.5 * 5.6

CYP4501AI-like mRNA

and

~~~~

3.02 f 0.93

4.05 i 1.26

1.25*0.61

Metabolism of BaP’

TABLE 2 Antioxidant Enzyme Activities, Mixed-Function Oxygenase (MFO) System Components and Activities, mRNA in Digestive Gland of Mussel (M. galloprovinciulis) from Sites in the Adriatic Sea and Venice Lagoona __~ ~~~~

“Chemical contaminants: single samples taken, average value for year given plus seasonal range in parenthesis; biochemical variables: means f SEM (n= 5 to 6) for April 1992 (SOD, catalase, P450, ‘41%peak’) or June 1992 (GST, CYP4501Al-like mRNA, BaP metabolism) samrles, *p < 0.05 comparing C.V.E. with Platform site. Unresolved complex mixture - aliphatic hydrocarbons in pg g-’ dry wt. ’ ng g-r dry, wt. d superoxide dismutase activity in SOD U g-’ wet wt. e mm01 g-’ wet wt. f pm01 rng-’ protein. g Arbitary units. h glutathione S-transferase activity in pmol min-’ g-’ wet wt. i Benzo[a]pyrene metabolism to free metabolites in pmol min.’ mg-’ protein.

C.V.E.

Crevan

Platform

~.

Site

Contaminant Levels, CYP450lAl-like

4 E 7

4

?J b 2: $

P

Impact of organic pollutants on goby

239

both goby and mussel, possibly reflecting variation in discharge and metabolism of contaminants, but most likely due to changes in tissue lipid levels (seasonal contaminant variation was less in skeletal muscle of goby - data not shown) (Walker & Livingstone, 1992). Contaminants were generally highest in both goby and mussel at the industrial sites of (respectively) Porto Max&era and C.V.E. Levels of PCBs and PAHs in mussel were similar at Crevan (Venice lagoon) and Platform site (Adriatic Sea), but aliphatic hydrocarbons (UCM - unresolved complex mixture) were higher in the former. Most biochemical measurements varied with sex (goby - data not shown) and season (goby and mussel). Hepatic EROD activity in both male and female goby was higher at Porto Marghera than at Crevan (3-57 times higher) and, to a lesser extent, also higher at Lio Grande than at Crevan (up to 5 times). A similar pattern was observed for levels of CYPlA protein, viz. for selected samples from June in arbitrary units (n= 3) 2.76 kO.71 (Port0 Marghera), 0.64~tO.22 (Lio Grande) and 0.01 *O.Ol (Crevan) (p < 0.05). The putative induction of CYPlA at Port0 Marghera is therefore consistent with the higher levels of tissue contaminants in these fish and indicative that contaminant-impact poses a threat to the health of these and other organism at the industrialised site. Catalase and putative DT-diaphorase (resorufin reductase activity) were higher in male but not female goby at Porto Marghera than at Crevan, but no differences were observed in SOD activity. More variation in resorufin reductase activity was seen at Lio Grande (see seasonal range - Table 1) which at times was as high as at Porto Marghera. The results therefore indicate some potential, but the need for more studies, for antioxidant enzymes as biomarkers of contaminant- or other-mediated oxidative stress (Livingstone ef al., 1992; Walker & Livingstone, 1992); in particular the need to characterize the properties and regulation of DT-diaphorase which in mammals is part of the [Ah] gene battery, along with CYPlAl (Nebert et al., 1990). Despite reported success with some biochemical measurements in bivalves (BaP hydroxylase, P450 and ‘418-peak’, antioxidant enzymes) (Livingstone, 1988; Garrigues et al., 1990; Porte et al., 1991), no single one has yet emerged as a widely used biomarker for organic pollution in molluscs, and a multi-parameter approach has been advocated (Livingstone, 1991). However, little or no consistent differences between sites were seen in mussels for SOD, catalase and GST activities, P450 and ‘418-peak’ levels (Table 2), NADPH-cytochrome c reductase activity, metabolism of BaP to putative protein adducts, and EROD-like activity (‘resorufin-fluorescence’ is bound to microsomes and removed by centrifugation) (data not shown). In contrast, levels of CYPlA-like mRNA and BaP metabolism to free metabolites (diols, diones, phenols) (Table 2) were higher or indicated to be higher in mussels in Venice lagoon (C.V.E. and Crevan) compared to the Adriatic Sea (Platform) (Table 2). Consistent with this, levels of CYPlA-like immunoreactive protein were also indicated to be higher at C.V.E. than at the Platform site, viz. in arbitrary units (n = 3) 10.85 f 3.80 c.f. 5.97 f 1.31. Correlation was therefore seen between these three measurements and levels of aliphatic hydrocarbons (UCM) but not PAHs and PCBs (main inducers of mammalian CYPlAl) (Table 2). The results are indicative of the existence of a CYPlA-like enzyme in mussel and argue for further study to investigate its biomarker potential.

D. R. Livingstone et al.

240

ACKNOWLEDGEMENTS This work was carried out in the framework of the UNESCO Project ‘Venice Lagoon Ecosystem’, a part of the Italian Ministerial Project ‘Venice Lagoon System’, from which it was financed. It was also supported in part by a European Environmental Research Organisation (EERO) postdoctoral fellowship to P.L.

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