Interaction of benzo a pyrene, hexachlorobiphenyl ...

BIOMARKERS

1997, 2, 153± 160

Interaction of benzo[a]pyrene, 2,3,3Â ,4,4Â ,5-hexachlorobiphenyl (PCB-156) and cadmium on biomarker responses in flounder (Platichthys flesus L.) Morten Sandvik, Jonny Beyer, Anders Goksù yr, Ketil Hylland, Eliann Egaas and Janneche Utne Skaare Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg± 1), 2,3,3Â ,4,4Â 5-hexachlorobiphenyl (PCB156, 2.5 mg kg± 1), and cadmium (cadmium, 1 mg kg ± 1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB-treated flounder after 2 and 8 days, respectively. The strongest induction (44-fold) was caused by BaP. No further induction was observed after additional treatment with PCB-156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre-treated with cadmium, whereas induction by PCB-156 appeared to be unaffected by pre-treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre-treatment with BaP and PCB prior to cadmium inhibited the MT induction (30± 50%) compared with cadmium alone. Furthermore, significantly higher glutathione S-transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB-156 prior to cadmium. GST selenium-independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.

Keywords: fish, biomarkers, PAH, PCB, cadmium, interactions, CYP1A, metallothionein.

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Introduction R elat io n sh ip s b etw een exp osure to env iro n m e n t a l con tam inan ts an d b io m ar k er re sp o n ses h av e b een w e ll do cu m en ted in f is h . Ind u ction o f C Y P 1 A in fish h as b een asso ciat ed w it h ex p o su re to p lan ar aro m a tic co m p o u n ds, su c h as p o l y aro m ati c h y d ro car b on s (PAH s) , p o lych lo r in ated bip h eny ls (P C B s), an d d io x in s (S teg em an and H ahn 199 4, B uc h eli an d F en t 1 9 95 , G o ksù y r 1 99 5 ). H en ce , bo th in th e field and i n th e lab orato ry, C Y P 1 A ind u cti on h as fre q u en tly bee n em p lo yed as a bio m ark er fo r su ch po llu tan ts. M etallo thion ein (M T ) is th e m ost w id ely us ed b iom ark er fo r env iro n m e n t a l m etal con tam in at io n. In fish , ti ss ue lev els o f M T g en eral ly i n c re ase w ith in cre asing ex p o su re to n on -ess en tial m etals such as h ep at ic ca d m i u m an d m e rc u ry, bu t m ay also b e in du ce d by hig h co p p er and zin c co ncen tratio n s ( G eo rg e and O lss on 19 9 4) . P ol lu ted are as are g en erall y ch aract erized b y a m ixtu re o f com p o u n ds, r ath er than b y si n gle ch em icals, w h i ch m ay re s u l t in eith er sy nerg isti c o r anta go nisti c eff ec ts o n aq u at ic o rg an ism s. T h eref o re, in fo rm ati o n re g ard ing su ch in ter actio n eff ect s clea rly is im po rta n t for a pro pe r in t erp ret ati o n of po llut an t- d ep en d en t b io m ark er re sp on ses re co rd ed in f ish fro m p o ll u ted a re as. T he object ive o f th e presen t stud y w as to ev alu ate CY P 1A and MT as bio m ar ke rs in fish in m ix ed ex po sure situ at io ns. T he inter active effects of BaP, PC B-15 6 and cadm ium were stu died b y seq ue nt ial adm in istr at io n of the chem ica ls in p ai rs co m pare d w ith adm inist ra tio n of th e chem ical s alo n e. E uro p ea n flo und er (P la tic hth ys flesu s) w as use d as m o d el sp ec ies in th e st ud y. T hi s e u ry hal ine flatf ish oc cur s in coastal an d estua ri ne reg io ns t h ro ug h ou t E urop e, and h as th erefore fre qu en tl y b een use d for the m o nito ri ng o f pol lut ant lev els an d eff ects in fiel d stud ies (v on West ern h ag en et al. 1981 , Stegem an et al. 1988, Bey er et al. 1996a, E ggens et al. 19 96 , H y llan d et al. 1996).

MATERIALS AND METHODS Chemicals 7-Ethoxyresorufin, resorufin and benzo[a]pyrene (BaP) (min. 98%) were purchased from Sigma. PCB-156 was kindly provided by Åke Bergman, Wallenberg Laboratory, University of Stockholm, Sweden. CdCl2 was obtained from May and Baker Ltd, Dagenham, UK. Alkamuls EL-620 (Emulphor oil) was obtained from Rhône-Poulenc Chimie, Paris, France. All other chemicals used in preparation and analyses of samples were of analytical grade.

Animals The experiments were performed at the High-Technology Centre (HiB) in Bergen, in the laboratory facilities of the Industrial Laboratory. Gonadally-immature flounder

Morten Sandvik, Eliann Egaas, Janneche Utne Skaare (author for correspondence) are in the Department of Toxicology, Central Veterinary Laboratory, PO Box 8156 Dep., 0033 Oslo, Norway; Janneche Utne Skaare is also in the Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, PO Box 8146 Dep., 0033 Oslo, Norway; Jonny Beyer, Anders Goksù yr are in the Laboratory of Marine Molecular Biology (LMM), University of Bergen, N-5020 Bergen, Norway; and Ketil Hylland is at the Norwegian Institute of Water Research, N-0411 Oslo.

(91± 376 g) were collected nearshore at Sotra, west of Bergen, Norway. Prior to the experiments, they were acclimated for at least 2 weeks, and fed every second day with commercial flatfish pellet. The feeding was stopped 3 days before the initial injections, and the fish fasted throughout the rest of the experiment. The water temperature was 8.5± 9.2 °C, and the salinity was 33.3± 34.5 psu during the experimental period. The fish were kept in 500 l tanks, containing a layer of shell sand.

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Treatment

Statistical methods

During treatment the fish were randomly selected from the main fish pool, but

Sample data were log transformed when necessary in order to allow the use of

individuals with fin injuries or other visible abnormalities were not included in the

parametric statistical methods. Parametric tests were always preceded by

experiments. BaP and PCB were dissolved in 50:50 acetone and Alkamuls, whereas

Bartlett’s test for homogeneity of variance. Student’s t-test was used to test the

CdCl2 was dissolved in 50:50 filtered seawater and Alkamuls. The toxicants or

difference between control and exposed groups. One-way ANOVA and

vehicle (controls) were administered to the fish by subcutaneous injections (1 ml

Tukey± Kramer HSD (multiple comparison) tests were used when differences

kg± 1). At the start, each flounder received a single injection with either PCB-156

between means of more than two groups were evaluated. The level of statistical

(2.5 mg kg± 1), BaP (2.5 mg kg± 1), cadmium (1 mg kg± 1) or the corresponding

significance for rejecting H0: `No difference’ between groups was set to p < 0.05.

vehicles. Two days post-injection, the BaP-treated flounder were either sacrificed or re-injected with each of the other toxicants. Similarly, PCB-treated fish were either sacrificed or re-injected after 8 days, whereas cadmium-treated fish were either sacrificed or re-injected after 15 days. The groups subjected to mixed exposure

Results

regimes of BaP and PCB, cadmium and BaP, or cadmium and PCB were sacrificed

General observations

10, 17, or 23 days after the initial injection, respectively. Samples were obtained

L iv er so m atic in dex , co n d itio n fact or an d d iff ere n t sizes w ere eq u ally d ist r ibu ted b et w een th e g ro up s, bu t th e av er ag e w eig ht fo r fem ale s w as si gni fica ntly h ig her th an for th e m al es. A naly si s o f co v arian ce ( D rap er and S m ith 1 9 81 ) sh o w ed th at b io m ar k er re sp o ns es w ere no t sig n if ican tl y aff ect ed b y se x. A p p rox im ate ly 5 % o f th e floun der die d du ring the ac cli m at io n per io d. Most o f the m o rta li ti es w ere p re sum ab ly du e to tai l fin inju ri es. S im ilar tail fin in jur ies w ere o b serv e d in so m e in d iv idu als du r in g th e ex po su re p erio d . H o w ev er, su ch in d iv idu als w ere ex cl u de d, sin ce ph y sio log ical stress of the finin ju ry co ul d hav e in fluen ced th e re sp o n ses m easure d. O ve ra ll, le ss th an 3% of th e tre ated fl ou nd er (4 o f 1 44 ) di ed dur in g th e ex po su re pe ri od. Int erest ing ly, th ree ou t of fo ur fish d ie d in th e PC B-exp osed g ro up re -injec ted w it h cad m iu m , w here as no m o rtal ity occu rre d in th e g ro up in je cte d w ith cadm iu m befo re PCB-156.

from a total of 114 fish. Six treated and six corresponding controls were sacrificed at each sampling point. Before sampling, the size of the fish, and external lesions were recorded. Damaged fish were not used.

Preparation and analyses of samples The gall bladder was carefully excised and the bile was frozen at ± 20 °C. Samples of liver for chemical analyses and hepatic subcellular samples (microsomal and cytosol fractions) were prepared as described in Beyer et al. (1997a) and frozen at ± 80 °C. Analyses of total protein were performed according to Bradford (1976) using bovine serum albumin as standard. Fluorescent biliary compounds of BaP (BaP± FACs) were measured by direct fluorometry as described by Beyer et al. (1997a). PCB-156 were measured according to Bernhoft and Skaare (1994). The detection limit of PCB-156 was determined in samples from both PCB-treated and untreated flounder. The detection limit was 44 and 0.88 ng g± 1 w.w. respectively, due to different dilution of the samples. The Environmental Toxicology Laboratory at the Norwegian College of Veterinary Medicine, Oslo,

Tissue levels of chemicals

Norway has participated in all four phases of the ICES/IOC/OCSPARCOM

T h e leve ls o f bil ia ry BaP-FAC s, he p ati c P C B -15 6, an d hep atic cad m iu m fo llo w in g su bcu tan eo u s ad m inistratio n o f the to x ica nts alon e o r in d iff ere n t p airs are sh o w n in Tab le 1. N o a p p a re n t i n t erfe re n ce in the accu m u lation o f th e ch em icals w as o b se rv ed w h en gro u p s w e re co m p a re d using o n e-w ay A N O VA. Regre ss io n an aly sis sho w ed th at b iliary BaP ±FA C lev els w ere p osi tiv ely co rre lat ed bo th to E R O D act iv ity an d C Y P 1A levels o n ly w hen B aP w as adm in is tered alo ne. N o co rr elatio n w as ob se rv ed betw een h ep atic P C B levels and C Y P 1A ind u ctio n . T h e M T ind uctio n w as no t p o si tively co rr elat ed to cad m iu m ex p o su re ei th er.

intercomparison exercise on the analyses of PCBs, and the analytical quality has proven acceptable. Liver samples for metal analyses were digested in concentrated nitric acid (Ultrapure, Merck) at 165 °C for 8 h, H2O2 subsequently added, and further digested at 140 °C for 2 h, according to the B. Welz protocol of atomic absorption spectroscopy (Weltz 1985). Cadmium was measured by graphite furnace atomization (Varian SpectrAA 400). The detection limit was 0.01 m g g± 1 w.w. A control system with regular analyses of reference materials was adopted, and measurements of these samples (NRCC Dogfish DOLT-1 and DORM-1) were within acceptable limits. CYP1A protein in hepatic microsomal samples was measured according to Goksù yr (1991) with the use of enzyme-linked immunosorbent assay (ELISA) and

CYP1A

rabbit-anti cod CYP1A IgG antiserum. CYP1A-dependent 7-ethoxyresorufin O-

In ter ac tio ns o n the b io m ar ker eff ects fo llow ing ad m inist ration o f th e to xic an ts alon e or in d iff ere n t p air s w ere in vestigated on tim ep oin ts d eriv ed fro m ea rlier tim e-cou rse st ud ies (F igure 1) . In BaP -tre ated f lo u n d er, a si gn ifica n t in d u ct io n of E R O D ac tiv it y an d C Y P 1 A p rot ei n lev el s (44 - an d -2 fo ld re lati ve to co n t ro ls, res p ect ively ) w as ob se rv ed 2 d ays after th e ad m in ist rat ion o f B aP (F ig u re s 2 an d 3 ). A si m il ar re sp o n se to B aP w as o b serv ed in th e g ro u p p re t rea te d w ith P C B -1 5 6 8 day s ea rli er (F igu re 2) . In th e gro u p re ceiv in g B aP fo llow ing cad m ium pre -tre atm en t 1 5 day s earlier, h o w ever, th e C Y P1 A ind uct ion (E R O D 3- fol d of co n t ro l) ap p eare d t o b e so m ew h at in h ib ited b y th e cad m iu m p re -tre at m ent. T h e C Y P 1 A in d uctio n in this g ro u p a p p e are d to b e lo w er th an in th e o th er B aP - ex p o sed g ro u ps, an d n ei th er th e

deethylase (EROD) activity was measured according to Stagg and Addison (1995) with resorufin as internal standard, assay temperature 20 °C, and assay pH 7.4. A control system of the CYP1A measurements was adopted with regular analyses of reference microsome samples from untreated and b -naphthoflavone treated cod. Hepatic metallothionein (MT) in hepatic cytosol was determined by pulse polarography as described by Olafson and Olsson (1991). Purified flounder MT was used as a standard. The MT concentrations in the standards were quantified by measuring the content of cysteine. Hepatic cytosolic GST activities towards CDNB (1-chloro-2,4-dinitro-benzene), ETHA (ethacrynic acid) and CU (cumene hydroperoxide) was measured according to Habig et al. (1974). The assay conditions for flounder have been described earlier by Egaas et al. (1993).

Interactive effects of pollutants in flounder

Treatment (day) None BaP (0) PCB-156 (0) Cadmium (0) PCB-156 (0)+BaP (8) BaP (0)+PCB-156 (2) Cadmium (0)+BaP (15) BaP (0)+cadmium (2) PCB-156 (0)+cadmium (8) Cadmium (0)+PCB-156 (15)

Sampling day 0 2 8 15 10 10 17 17 23 23

155

(m g BaP ml ) ± 1

Tissue levels

(m g PCB-156 g± 1)

2.7 ± 1.1 (6) 256 ± 34 (5)

0.001a

194 ± 45 (6) 157 ± 39 (5) 214 ± 26 (6) 401 ± 230 (5)

4.8 ± 1.6 (6) 4.1 ± 1.8 (6)

4.3 ± 1.1 (6)

7.2 ± 0.4 (3) 9.2 ± 2.7 (5)

(m g Cd g± 1) Not analysed

32 ± 5 (6)

24 ± 2 (6) 25 ± 5 (6) 17 ± 3 (3) 25 ± 6 (5)

Table 1. Hepatic levels of PCB-156 and cadmium and biliary BaP levels in flounder (Platichthys flesus) following subcutaneous administration of the toxicants alone or in different sequential combinations.

Each flounder received a single subcutaneous injection with either PCB-156 (2.5 mg kg ± 1), BaP (2.5 mg kg ± 1) or cadmium (1 mg kg± 1) at the start of the experiment. BaP-treated fish were either sacrificed or re-injected with one of the other toxicants 2 days post-injection. PCB-treated fish were either sacrificed or re-injected with one of the other toxicants 8 days post-injection. Cadmium-treated fish were either sacrificed or re-injected with one of the other toxicants 15 days post-injection. Flounder treated with BaP and PCB-156 were sacrificed 10 days after the first injection. Flounder treated with cadmium and BaP were sacrificed 17 days after the first injection, whereas fish treated with cadmium and PCB-156 were sacrificed after 23 days. Values are presented as mean±SEM (n). a

Detected in only one control fish.

E R OD nor C YP 1A p ro tein lev els w ere sig n ific ant ly d iff ere n t fro m the corr esp o n d i ng c o n tro l g ro u p lev els. H o w ev er, th is a p p a re nt inh ibit o ry eff ect o f cad m iu m w as n o t stat ist ical ly sig nificant w hen th e g ro u p s w e re c o m p a re d u si n g o n e- w ay A N O VA . In PCB-156-tr eat ed flo un d er, a si g n if ican t in d u cti o n o f E RO D act iv it y an d C Y P 1A pro te in lev el s (3 - an d 1 .5 -fo ld of

Figure 1. Temporal hepatic CYP1A induction (EROD activity) following intramuscular administration to BaP (l), PCB-156 (n), and induction of hepatic MT levels (s) following cadmium exposure to flounder. H: Significantly different from corresponding control group as determined with Student’s t test. Adapted from Beyer et al. (1996a).

co n t ro ls, res p ect ively ) w as o bse rv ed 8 da y s af ter th e P CB inj ect io n (F igu res 2 an d 3, re sp ect ivel y ). M ore or less sim ila r re sp o nses to th e P C B ex p o su re w e re re c o rd ed i n th e tw o gro u p s p re -t re at ed w ith eith er B aP 2 d ays earlier o r cadm iu m 5 d ay s earlier. T hu s, CY P 1 A ind u ct io n in P C B -tre ated fish , w as n o t aff ec ted by B aP or cad m ium pre- tre at m en t. I n cad m iu m -tre a ted fl ou n d er, no C Y P 1A in d u ction w as re co rd ed in th e gro u p s p re -t rea te d w ith B aP o r P CB , as c o m p are d w it h th e re sp ectiv e con t rol s (F igu re s 2 an d 3).

Figure 2. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) in flounder following subcutaneous administration of BaP, PCB-156 or cadmium, either singly or in different combinations. Treated fish are represented by closed bars (means±SEM (6)). Open bars represent corresponding controls injected with the vehicles only. H: Significantly different from corresponding control group as determined with Student’s t test. Treatment: BaP: Administration at day 0 and sampling at day 2. PCB-156+BaP: Administration of PCB-156 at day 0, administration of BaP at day 8 and sampling at day 10. Cd+BaP: Administration of cadmium at day 0, administration of BaP at day 15 and sampling at day 17. PCB-156: Administration at day 0, and sampling at day 8. BaP+PCB-156: Administration of BaP at day 0, administration of PCB-156 at day 2 and sampling at day 10. Cd+PCB-156: Administration of cadmium at day 0, administration of PCB-156 at day 15 and sampling at day 23. Cd: Administration at day 0 and sampling at day 15. BaP+Cd: Administration of BaP at day 0, administration of cadmium at day 2 and sampling at day 17. PCB-156+Cd: Administration of PCB-156 at day 0, administration of cadmium at day 8 and sampling at day 23.

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Figure 3. Hepatic CYP1A protein levels in flounder following subcutaneous administration of BaP, PCB-156 or cadmium, either singly or in different combinations. Treated fish are represented by closed bars (means±SEM (6)). Open bars represents corresponding controls injected with the vehicles only. H: Significantly different from corresponding control group as determined with Student’s t test. Treatment: BaP: Administration at day 0 and sampling at day 2. PCB-156+BaP: Administration of PCB-156 at day 0, administration of BaP at day 8 and sampling at day 10. Cd+BaP: Administration of cadmium at day 0, administration of BaP at day 15 and sampling at day 17. PCB-156: Administration at day 0, and sampling at day 8. BaP+PCB-156: Administration of BaP at day 0, administration of PCB-156 at day 2 and sampling at day 10. Cd+PCB-156: Administration of cadmium at day 0, administration of PCB-156 at day 15 and sampling at day 23. Cd: Administration at day 0 and sampling at day 15. BaP+Cd: Administration of BaP at day 0, administration of cadmium at day 2 and sampling at day 17. PCB-156+Cd: Administration of PCB-156 at day 0, administration of cadmium at day 8 and sampling at day 23.

Figure 4. Hepatic metallothionein (MT) levels in flounder following subcutaneous administration of BaP, PCB-156 or cadmium, either singly or in different combinations. Treated fish are represented by closed bars (means±SEM (6)). Open bars represents corresponding controls injected with the vehicles only. H: Significantly different from corresponding control group as determined with Student’s t test. Treatment: BaP: Administration at day 0 and sampling at day 2. PCB-156+BaP: Administration of PCB-156 at day 0, administration of BaP at day 8 and sampling at day 10. Cd+BaP: Administration of cadmium at day 0, administration of BaP at day 15 and sampling at day 17. PCB-156: Administration at day 0, and sampling at day 8. BaP+PCB-156: Administration of BaP at day 0, administration of PCB-156 at day 2 and sampling at day 10. Cd+PCB-156: Administration of cadmium at day 0, administration of PCB-156 at day 15 and sampling at day 23. Cd: Administration at day 0 and sampling at day 15. BaP+Cd: Administration of BaP at day 0, administration of cadmium at day 2 and sampling at day 17. PCB-156+Cd: Administration of PCB-156 at day 0, administration of cadmium at day 8 and sampling at day 23.

MT A dm in ist r ation of ca d m iu m alo n e cau se d si g n ifican tly ele v at ed co n cen tr atio n s o f hep at ic M T (2 .5 -fo ld) 1 5 d ay s af ter inje ct ion (F ig ure 4) . A sig nific ant re d ucti o n (3 7 % ) in M T lev els w as o b se rv e d w hen B aP w as in ject ed p r io r t o cad m iu m , as co m p are d w it h t re atm en t w ith cad m iu m alo n e, an d t h e M T lev el w as n o t si gn if ic an tly d iff ere nt fro m th e co rr esp o n d in g co n trol g ro u p (F ig u re 4) . S im ilar ly, f lo u n d er ex p o sed to cad m iu m 1 5 d ay s p rior to re- in je ctio n w it h B aP h ad 3 0 % lo w er M T lev els th an in f ish in jec ted w ith cad m iu m alo n e (F ig u re 4 ). In the gro u p p re- tre ated w ith P C B -1 5 6 an d th en in jected w ith cadm iu m , th e M T lev el w as si g n ifican tly lo w er (5 0 % ) than in th e gro u p re ceiv i ng cad m iu m o n ly, b u t th e le v els w ere st il l si gni fic an tly el ev ate d (2 .5 -fo ld ) as com p are d w it h t h e c orr esp on d in g co n tro l gro u p ( F ig u re 4) . T h e sa m e rel ativ e in du ct ion w as a lso o b se rv e d w h en c ad m i u m w a s a d m in ist ere d b ef ore P CB-1 56 (F igure 4). T he m ixed tre atm en t w ith P C B an d B aP ap p ea re d to d ep re s s the M T lev el w hen P C B w as ad m in istered p rio r to BaP (F igu re 4 ) , b ut not w h en th e tox ican ts w ere ad m in i stere d in th e rev er se ord er (F ig ure 4 ). H ow ev er, n ei th er BaP n o r P C B-1 56 a p p e a re d to in fluen ce the M T re sp o n se w h e n th ese ch em i cals w e re ad m in istere d alo n e (F ig u re 4).

acet o n e: A lkam u ls (co ntro ls fo r B aP an d P CB inj ect io ns) af ter fil tere d se aw ater:A lk am uls (con tro ls for ca d m iu m in jec tio n), w e re h ig he r (si g n if ican t at day 23 ), th an in co ntro ls in jected w it h th e sa m e v eh icles in rev er se o rd er (re su lt s n o t sho w n ) . T h e selen ium - in d ep en d en t G S T pero xid as e act ivity tow ard s C U ap p eare d to b e u n aff ect ed b y an y tre atm en t i n the p re s en t st ud y (F igu re 5).

Discussion Exposure regime In p re v io u s st ud ies w ith flo un d er g iven in tr am u scu lar in je cti o ns, o cc asi o nal ly a sm alle r fracti o n o f the inject ed do se w as lea k in g o u t o f the injectio n sp ot. S u bcu taneo u s ad m inistratio n seem ed to solv e th is p ro b l em an d w as c h o sen as an al tern ativ e exp o su re reg im e. H ep ati c levels o f ca d m ium an d b iliary le ve ls of B aP w ere si m ila r fo llo w ing b oth ro utes o f e x p o su re. L ev els o f P CB -1 5 6 af ter 8 da ys, ho w ev er, app eare d to b e lo w er fol low in g sub cu tan eo us r at h er th an in t ram u scu lar ad m inist r atio n (4 .3‰1 .1 an d 18 .6‰2 .7 m g g ±1 liv er w. w., re sp ec tiv el y).

GST

CYP1A

H epati c gluta th io ne S - tr an sf erase act iv it ies to w ard s C D N B an d E T H A w ere sig n ifi can tl y el ev ate d (2 -fo ld ) in flo u nd er a dm in i ste re d cad m ium , eith er alo n e o r fo llo w in g p ret re atm en t w it h B aP or P C B-1 5 6 (F ig u re 5 ). H o w eve r, in d u cti o n w as ab o l ish ed w h en ca dm i u m w as ad m in ist ere d p rio r to B aP o r P C B-15 6. GS T acti vi tie s in co nt ro ls in jected w ith

T h e p re sen t stud y dem o n strated th at C Y P 1 A in f lo un der w as re sp o n si v e to tre at m en t w ith b o th BaP an d P C B -1 56 . T h e d iff ere n ces in tem p o ral ind u cti o n pat tern s fo llow in g tre a tm e n t w it h BaP o r P C B -15 6 h av e bee n est ab li sh ed ea rli er (B ey er et a l. 1 9 97 b ), w h ere the ob serv e d m a x i m um i n d u cti o n oc cu rre d 2 an d 8 d ays after in tr am usc ular in je ctio ns w ith B aP and P C B -


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Figure 5. Hepatic glutathione-S-transferase (GST) activities towards 1-chloro-2,4 dinitrobenzene (CDNB) (A), ethacrynic acid (ETHA) (B) and GST selenium independent peroxidase activities towards cumene hydroperoxide (CU) (C) following subcutaneous administration of cadmium alone (l), injection of BaP 2 days prior to cadmium injection (n) or injection of PCB-156 8 days prior to cadmium injection (s). The flounder were sacrificed 15 days after the cadmium treatment. Open symbols represents corresponding controls injected with the vehicles only. Values are means±SEM (6). H: Significantly different from corresponding control group as determined with Student’s t test.

respecti vely. T he re su lts are g en er ally in acco rd a n ce w i t h p re vious st u dies (S k aa re et a l. 1 9 91 , Bern h o ft an d S k aare 1 994 , L evine et al. 19 9 4, v an der Wei d e n et a l. 19 94 ). T h e P C B co ng en er used in th e presen t stu dy (2 ,3,3 Â,4 ,4Â,5- PC B, IU PA C no . 1 56) is a m on o-o rt h o su b st it u ted ana lo g ue o f P C B- 12 6 , one of th e ultim at e to x ic P CB s in the n o n -o rt h o cl ass . T h e si n g le ex tra ort ho-chlo rin e o f P CB -1 5 6 lo w er s b o th th e to x ici ty an d C Y P 1A -indu cing po tency co n sid erab ly as co m p are d w it h P C B-126 . H ow eve r, beca use m o n o -o rt h o PCBs (e.g. PCB-105, -118, and -15 6) o cc u r in h ighe r en v iro n m en tal co n c en t ra tio ns th an th eir no n -o rt h o co u n te r p art s, th eir en v iro n m e n t al i m p ac t i s co nsid ered to be h igh ly sig n ifi can t (Tana be 19 9 2, D e Voo gt e t al. 19 90 ). A less than add i tive eff ec t in th e CY P 1A re s p o n se w a s o b serv ed in th e m ix ed B aP ±P C B tre atm en t g ro u p s. W h en B aP w as in je cted pr io r to P C B , th e lack o f co n tr ib u tio n fro m B aP m ay b e exp lain ed b y th e sho rt d u ratio n o f C Y P 1 A in d u ction

fo llo w in g B aP ex p o su re , as w as o bserv ed i n t h e p re v i o u s tim e-cou rse st u d y w ith flo un d er (B ey er et a l. 1 9 96 b) . H o w ev e r, in th e sam e stu dy, th e C Y P 1A in duc tio n o f P C B -1 56 w a s m o re persi sten t. T h e C Y P 1A activity w as st ill si gn ific antl y elev ated 1 6 day s follo w in g P C B -1 56 tre a tm en t , b u t th e re w as n o a p p a re nt co n trib utio n f ro m the P C B t re a tm en t w h e n re -injected w ith B aP. T he C Y P 1 A level s in g ro u p s t rea ted w ith ei th er B aP o r P C B -1 56 , si ng ly o r in any co m b in at io n, w ere n ot si g nificantly d iff ere n t w h en c o m p are d u sin g on e- w ay A N O VA a n d Tu key ±K ram ers H S D test . T h is m ay in di cate th at e nzy m e sat u r atio n w as alre a dy re ac he d b y ad m in istr at io n of b o th co m p o u n d s a lo n e. I n th e p re sen t s tu d y, there w as n o in f lu ence o f cadm ium e x p o su re al on e o n th e CY P 1A in flo u nd er, bu t E R O D indu cti on b y B aP w as si g nificantl y su p p re sse d b y prio r ex po su re to ca d m iu m , w h e rea s CY P 1A p ro tei n lev els re m a i n e d u n c h an g e d .

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Inh ibi ti on of CY P 1A b y en vi ro n m en tal ch em i cals su ch a s som e P C B s an d m etals o n C Y P 1 A h as b een d em o n strated earlie r in fish (F en t an d S teg em an 1 9 9 3 , S teg em an an d H ah n 1 994) . Both CY P 1A leve ls an d asso cia ted ca tal yt ic act iv iti es st ro ng ly decre ase d fo llo w ing ad m in ist ratio n o f trib u ty ltin (F en t and S teg em an 1 99 3) an d cad m iu m ( G eo rg e an d Yo u n g 1 98 6, G eo rg e 1 98 9 ). T h e in h ib it io n cau se d b y cad m iu m w as d ose-depend ent, at 1 m g k g ±1 a 9 0 % d ecre ase w as o b serv ed i n p laic e (P leu ro n ectes p la tess a). H o w ev er, in d u ct io n o f C Y P 1 A h as also been ob serv ed w it h cad m iu m in E u ro p e an ee l (A ng uill a a ng u illa) an d E u ro p ean se a b ass (D ic en tra rc h u s lab ra x) (L em aire -G o ny et al . 1 9 9 5 ). In se a b as s, ca d m iu m t re atm ent cau sed a 1 0-fo ld in crease in E RO D act iv it y. T h e co n tr adic tory n a tu re o f su ch o b se rv atio n s illu strates th e n eed for obtai ning kn ow le d g e ab o u t th e sp eci fic sp ecies to b e u se d in m on itor in g. T h e ab o lish ed C Y P 1 A in d u ctio n o f 3 -M C b y coin ject ion w ith ca d m i u m in p laice su g g ested th at th is w as d u e to a d ecre ase i n e n zy m e p ro tein rath er th an d ire c t inh ibit ion o f act iv it y b y cad m iu m (G eo rg e 19 89 ). F u rt h e rm o re , M ea n s et al. (19 7 9) hav e sh o w n th at th e d ecrea se in C Y P p ro tein lev els in cad m iu m -tre ated r at s w as d u e to b o th a d e c re ase in p ro tein sy n the si s an d in cre ase d d eg r ad at io n o f th e C Y P enzym e by a h ae m ox y g en ase . CY P 1A ind u ctio n by P C B -15 6 ap p ea re d to b e u n aff ec ted by p re- tre a tm en t w i th cad m iu m . T h e m ec h an ism s res p o n sib le fo r this d iffe re nce are u nk n o w n . H a lo g en ated aro m at ic h y d roc arbon s, su ch as P C Bs, are slo w ly m etab o lized an d they continue to in du ce sy n th es is o f C Y P 1 A m R N A an d p ro te i n o ver tim e (H ah n and S teg em an 1 9 9 4 ), w h ile re ad i ly m etab olized in du cers lik e B aP d o n o t p ro d u ce co n t i n u e d transcr ip tio n al act ivation as th ey are m etab o liz ed . O th er m ec h an ism s su ch a s di ffe re n t ial p ro tein st ab iliza tio n o r enhanced tran slatio n o f a m in o r m R N A p o o l, m ig h t co n trib u te to p er si st en ce of C Y P 1 A (K lo ep p er- S am s an d S te g em an 1 9 94 ).

GST H epat ic glu tath ion e S -transf erase ac tivi ties to w ard s C D N B an d E T H A in co n tro l fl ou n d er w ere in the ran g e re p o rte d b y G eo rg e (19 89 ). T h e re su lt s in dicat ed th at B aP an d P C B -1 5 6 had no eff ect o n g luta th io ne S -tran sf eras e act iv iti es in flo u nder. Treat m en t w i th 3 - m eth y lch o la nth ren e (3-M C ) de pre sse d G S T ac tiv itie s in flou nd er (S co tt et a l. 19 9 2) , w h ere as n o eff ect w as seen in F ath ead m in n o w s an d S h eep sh ead m in no w s (Jam e s e t al . 1 98 8 ). In co nt rast , in d uct ion b y 3-M C has b een o b se rv e d in p la ic e (G eo rg e an d Youn g 1 98 6 ). S im ilar inte rsp ec ies v ariatio n s h ave b een o b serv ed fo llo w ing tre at m en t w i th c o m m e rci al P C B m ixtu re s such as A ro chlo r 1 2 5 4 an d C lo ph en A 5 0 (A n der ss o n et a l. 19 8 5, S co tt et a l. 1 99 2 ). It is po ssib le, as in m am m als, that C Y P 1 A in du cers h av e d iff ere n t eff ect s on v ar io u s GS T iso form s. C D N B ac ti v it y is an in te g rat io n of th e ac ti vi ty of n ear ly all o f th e d iff ere n t G S T iso en zy m es an d do es n o t p ro v id e in fo rm ation o n the iso fo rm co m p o siti o n . Q uan tifica tio n o f the variou s G S T su bu nits u si n g h ig h p e rf orm a n c e l iq u i d ch ro m ato g r ap hy h as p ro v e n m o re in fo rm ative as re g ard s is o fo rm co m p o si tio n th an eith er activity m e as u re m en t s o r im m u n o h ist o ch em i stry ( O st lu n d F arr an ts e t al . 1 98 7, P aro la et al . 19 93 , E gaas et al . 19 94) . G S T act ivi tie s to w ard s C D N B and E T H A w ere si g ni fican tly elev ated in fl ou n der ad m in istere d cad m iu m , eith er alo ne o r fo llo w in g p re -t rea tm en t w ith B aP o r P C B -1 5 6. In trap erit on ea l co injectio n o f B aP an d cad m ium pro du ced a sign if icant i n c rea se in G S T ac ti vit ies in b lac k se a b as s (C entro p ri stis st ri at a), w h ere as t h e i nd uc tio n w as ab oli sh e d w hen cad m iu m w as inje ct ed p rio r to B aP (F ai r 1 98 6 ). In ou r st u dy, th e i n d u c ti o n w as su p p re sse d w h en c ad m iu m w as ad m i n iste re d pr ior to B aP or P CB-156 . G S T act iv iti es in con tro ls in jected w ith ac et on e:A lk am uls af ter filt ere d seaw ater:A lk am u ls, w ere h igh er (si g n ific an t at day 2 3 ) th an in con tro ls injected w ith the sa m e v eh icles in re v erse o rd e r, su g gest ing a po ss ib le ac et on e eff ect on CD N B ac ti v it y in flo u nde r.

Metallothionein T h e pres en tl y o bse rv ed M T in d u ct io n in flo u n de r fol lo w in g inj ec tio n of ca dm iu m al on e con firm s ear lier st u d ie s w ith o th er tel eo st sp eci es , w h ic h h av e de m o n st rat ed th at th e h ep ati c M T lev el is indu ced (in a d ose- d ep end ent m ann er) fo llo w ing m eta l ex p o su re (R och and M cCarter 1984, G eorge 19 89, H og str an d an d Haux 1 99 0 ). F urt h erm o re, th e hep at ic M T lev el in fi sh appe ar s no t to b e in d uce d by en v iro n m en ta l p o llu tan ts o th er tha n m et als (O vern el l an d A b d u llah 1 9 8 8 , S ul aim an et al. 19 91). A s i n th e p re sen t stu d y, w h e re b ot h BaP an d P C B -15 6 p ret re at m e nt ex ert ed an in h ib ito ry eff ect u p o n th e M T i n d u ction respo n se to w a rd s cad m iu m , i n h ib ito ry in flu en ce s o f m ix ed e x p o su re re g im es on fish M T re sp o n se s h av e b ee n re co rd e d b y se veral other inv es ti gato r s. F o r ex am p le, in p la ice (P leu ro n ectes pla tessa L .), Georg e an d You n g (1 98 6 ) in jec te d a m ix t u re of 3-m eth y lch o lan th re n e an d c ad m i u m , an d f o u n d that indu ctio n of hep at ic M T w as d elay ed 1 w eek co m p are d w it h w hen cad m iu m w a s ad m i n i st r ated alo n e . Inhibition o f M T ind uctio n , as p re se n tly o b se rv e d w h e n P C B w as injec ted p rior to cad m iu m , is lik ely to in cre ase th e m eta l to xicity. S uch in ter ac tio n eff ects m ay ex p lain th e m o rt al ity ob se rv ed in this tre atm en t g ro u p .

Summary G iv en th e w id espre ad p ollu tion o f th e m arin e en v iro n m en t b y m i x t u res of b ot h org anic p o llu tan ts an d h eav y m etals, and th eir p o tential to m o d if y th e f un ction an d ind u ct io n of b io m ark ers, f u rt h er st u d ies o f p ollu tant in teract ion eff ects sho u ld b e co nd u cted in f ish. T h e p re se n t in ter actio n st udy w ith flo u n der h as in v est ig at ed su ch eff ect s for thre e m o de l p o llu tan ts ( B aP, P C B -1 56 , ca d m iu m ) an d th re e b iom arkers (C Y P 1 A , m eta lloth ion ein an d G S T ). T h ese exp er im en ts hav e d em o n str ated th at chem i cal m ix tu re s m ay aff ec t b iom ark er re sp on ses d iff eren tly fro m c om p o u n ds g iv en al on e , an d t hat b o th C Y P 1A an d m etallo th io n ein are in flu en ced by p o llut ants o th er th an their p r im ary ind u cers. A lso , the seq u en ce o f e x p o su res m ay b e o f im po rt an ce for th e bio m ark er si g nal . A n u m b er o f oth er p o ll u tants ( an d b iom ark ers) are cand idates for si m ila r in v est ig atio n s. S u ch info rm atio n is in d ispen sible fo r th e ap plicati o n and im p lem entatio n o f fish b io m ark ers fo r p o llu tio n stud ies in n atu ral fish po p u latio n s in h abitin g p o llu ted co as tal are a s.


Acknowledgements This research was funded by the Norwegian Research Council, Program on Marine Pollution, Grant no. 295999. The authors would like to thank Dag Grù nningen, Christin Plassen, Vidar Berg (National Veterinary Institute, Oslo), Kjersti Anita Helgesen (University of Bergen), Jaran Strand Olsen (University of Oslo), and Nina O. Svendsen (The Norwegian Crop Research Institute, Ås) for assistance in preparation and analysis of samples.

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