Shiqian Zhu

Susan Codi King

Mary L Haasch
email: mlhaasch@olemiss.edu

Biomarker induction in tropical fish species on the Northwest Shelf of Australia by produced formation water

Keywords: CYP1A, CYP2K1, CYP2M1, produced formation water, tropical fish, Australia, biomarkers

Normal operation of oil well platforms results in the discharge of produced formation water (PFW). The expression of CYP1A, CYP2M1-and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A, and, in addition, CYP2K1/2M1-like proteins. In a subsequent caged-fish study in the same location a second species, Stripey seaperch (Lutjanus carponotatus), caught at a clean site, were distributed to three caging sites in a PFW gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T=0, T=3 and T=10 days. Significant increases of CYP1A, one CYP2K1-and two CYP2M1-like proteins were noted at Site A at T=10 d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T=3 d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1-and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and induction of enzymes in the CYP2 family suggest new biomarkers for PFW. In addition, the novel response of one CYP2K-like protein requires further verification, but offers promise for improved monitoring of sub-lethal responses in marine organisms.

Introduction

Produced formation water (PFW), the oily water usually discharged from a platform after separation from the oil, is made up from formation water (water associated with the oil in the reservoir) and potentially includes water which was injected into the reservoir to maintain pressure for oil production [START_REF] Holdway | The acute and chronic effects of wastes associated with offshore oil and gas production on temperate and tropical marine ecological processes[END_REF]. As oil fields age, the amount of PFW increases as the reservoir fills with injected water [START_REF] Henderson | Potential impact of production chemicals on the toxicity of produced water discharges from North Sea oil platforms[END_REF].

When discharged, the PFW contains a large variety of naturally occurring materials from the formation, which include residual volatile and non-volatile hydrocarbons not removed by the separation regime, and also contains chemicals added to the production stream (Burns et al., 1999). Therefore, the composition of the discharged PFW is very complex. Almost all offshore oilfields produce formation water that can have significant environmental effects if not handled properly. Australia's oil and gas production activity is predominantly on the Northwest Shelf of Western Australia, off the continental margins between the North West Cape and Dampier [START_REF] Swan | Environmental Implications of Offshore Oil & Gas Development in Australia: The findings of an independent scientific review[END_REF]. There has been excellent work on the toxicology of PFW [START_REF] Holdway | The acute and chronic effects of wastes associated with offshore oil and gas production on temperate and tropical marine ecological processes[END_REF][START_REF] Neff | Bioaccumulation in Marine Organisms: Effect of Contaminants from Oil Well Produced Water[END_REF][START_REF] Neff | Oil well produced water discharges to the North Sea. Part II: comparison of deployed mussels (Mytilus edulis) and the DREAM model to predict ecological risk[END_REF]. Unfortunately, little is known of the effects of PFW on the tropical marine environment, especially for tropical reef fish. One study demonstrated minor toxicity of PFW from Harriet A oil platform to corals and their symbiotic algae [START_REF] Jones | The effects of produced formation water (PFW) on coral and isolated symbiotic dinoflagellates of coral[END_REF]. Another study illustrated a preliminary toxicological assessment of PFW including analytical detection and acute toxicity bioassay using European sea bass larvae [START_REF] Mariani | Produced formation waters: a preliminary study on chemical characterization and acute toxicity by using fish larvae (Dicentrarchus labrax L., 1758)[END_REF]). An independent scientific review commissioned by the Australian Petroleum Production and Exploration Association (APPEA) and the Energy Resource Development Corporation (ERDC) highlighted the need to conduct environmental assessments of industry practices in shallow coastal ecosystems around Australia.

In 1998, the Australian Institute of Marine Science (AIMS) conducted a pilot study to evaluate the use of sub-lethal stress indicators in fish for assessing exposure to organic contaminants associated with PFW discharged from an oil and gas production platform on the Northwest Shelf of Australia. The pilot study results confirmed that within 1000 m from the Harriet A platform there was potential for biological effects in fish populations exposed to PFW effluent and that further investigation was warranted at this site (Codi King et al., 2005a). For this study, these samples were re-examined for CYP1A using more universally repeatable conditions, and, in addition, the response of CYP2K1/2M1-like proteins was determined. In May 2003, AIMS conducted a comprehensive biological and chemical assessment of PFW effluent at the Harriet A platform. This more comprehensive study utilized a controlled system in which fish from a reference site were collected and placed in cages, and the cages were located in a gradient line moving away from the point source of PFW being discharged at Harriet A. The work described here is a subset of the suite of biomarkers conducted for the 2003 study and presents in detail the cytochrome protein biomarkers in an ecologically relevant reef species, Stripey seapearch (Lutjanus carponotatus) for the evaluation of the potential effects of PFW discharge at the Harriet A Platform on the Northwest Shelf of Australia.

Materials and Methods

Pilot study: study site and fish collection

Harriet A, an oil and gas production platform located 135 km west of Karratha on the Northwest Shelf of Australia, was the main focus of this study (Figure 1). The hydrocarbon concentration of PFW effluent in the sea-surface micro-layer, water column, sediments and biota, at this location, have been well documented (Burns and Codi, 1999) The site selections for the pilot study have previously been described in Codi King et al. (2005a). Gold-spotted Trevally was chosen for the pilot study because of its importance both commercially and recreationally to the NW Shelf of Australia. A total of 41 Gold-spotted Trevally were collected from each of three sites:

Harriet A (n=23, Mean + SD; 3817 + 1532 g total weight, 654 + 103 mm total length, age 1-5 yr); Harriet C (n=6, Mean + SD; 3608 + 1503 g total weight, 654 + 103 mm total length, age 2-6 yr); and reference site off the Montebello Islands (n=12, Mean + SD; 3992 + 1122 g total weight, 660 + 75 mm total length, age 2-5 yr). The data demonstrated there were no significant differences in total weight, total length and age of fish between the three study sites (P < 0.05). All fish were processed as previously described in Codi King et al. (2005a). Briefly, livers were removed, frozen in liquid nitrogen and transported to AIMS via a liquid nitrogen dry shipper remaining stored in liquid nitrogen until thawed for microsomal preparation (see Section 2.3).

Caged fish study: Study site and fish collection

The PFW plume from Harriet A forms a fine surface slick that moves in a NNW direction on the ebb tides and SSE direction on the flood tide. The PFW hydrocarbons in the sea-surface microlayer were detectable to a distance of 1 km. The lighter MW components (naphthalene and homologs) were in the dissolved phase while the heavier MW hydrocarbons (phenanthrene and homologs) were associated with the particulate phase. The particulate phase was removed from the water column within a distance of 0.9 -2 km of Harriet A (Burns and Codi, 1999;[START_REF] Holdway | Tracking produced formation water discharge from a petroleum production platform to the North West shelf[END_REF]. Therefore for the caged fish study, the cages were situated along the gradient of PFW formed as the plume moves away from the discharge site. Site A (within 200 m NNW of Harriet A) was the near-field site; Site B (~ 1000 m NNW of Harriet A) was the far-field site and Site C (~20 km NNW of Harriet A; in the Montebello Islands) was chosen as the reference site due to the lack of known contamination (Figure 2). The field component of this project was conducted aboard the AIMS RV Cape Ferguson from May 16-31, 2003. Initially, the target species was Red Emperor (Lutjanus sebae), since it could be caged successfully (Mike Cappo, pers. comm.). On the first day of collection; however, only 5 juvenile L. sebae were caught and another species, Stripey seaperch (Lutjanus carponotatus) were being trapped at a much higher proportion (10:1). L. carponotatus is an important recreational fishery in Dampier Harbor and preliminary biomarker research with this species from the Harriet A pilot study [START_REF] Codi | Northwest Shelf of Australia: Assessment of sub-lethal stress indicators in fish as potential indicators of petroleum hydrocarbon exposure[END_REF] suggested that it would suffice as a substitute species for L. sebae. From May 16-19 2003, an estimated 130 L. carponotatus were collected by fish traps along the Dampier Harbor LNG pipeline and maintained in a 1000-L holding tank with running seawater and air. On May 21, all fish were placed in cages with 2 cages per station, 15 fish per cage at each of 3 sites (Figure 2). Fish were fed individually quick frozen (IQF) pilchards of the highest quality (Western Australia Bait Supply, Fremantle), collected from clean local waters off of Albany, WA. Food canisters were replenished every two days during the field exposure. Fish were sampled at T=0, 3 and 10 d.

A total of 50 Stripey seaperch were analyzed for CYP1A, CYP2M-and CYP2K-like proteins from the holding tank (T=0), and T=3 and T=10 for cage Sites A, B, and C. There were no significant differences for total weight, total length and age between the study sites (P < 0.05; Table 1). All fish were handled and processed in a similar manner as stated for the pilot study.

Microsomal preparation and Western blot

Hepatic microsomal samples were prepared at the Australian Institute of Marine Science, Townsville, Qld (AIMS) following methods previously described [START_REF] Haasch | Effects of vehicle, diet and gender on the expression of PMP70-and CYP2K1/2M1-like proteins in the mummichog[END_REF] and shipped on dry ice to The University of Mississippi, University, MS. Hepatic microsomal samples were stored at -80˚C until analyzed. Protein content of the microsomal fraction was determined by Bio-Rad DC Protein Assay (Hercules, CA) using bovine serum albumin (BSA) as a standard. Microsomal proteins (10 µg per well) were separated using pre-cast Criterion SDS-PAGE gels (10%T, 2.7%C resolving gel; 4%T, 2.7%C stacking gel; Bio-Rad) at 200V for 1 h with the Bio-Rad Criterion apparatus. Proteins were then electrotransferred (100V for 0.75 h) to a PVDF membrane (FluoroTrans W, 0.2 µm; VWR) together with one lane containing MagicMark protein standards (contain proteins with IgG binding site; Invitrogen, Carlsbad, CA) detectable by chemiluminescence, for molecular weight determination and for evaluation of transfer efficiency. Sample loading buffer also contained Pyronin Y which is visible on the membrane and marks the dye front. Detection of proteins of interest was accomplished by using one of the primary polyclonal antibodies; rabbit anti-fish CYP1A (two synthetic peptides used as immunogens;

BioSense, Bergen, NO distributed by Cayman Chemical, Ann Arbor, MI), rabbit anti-trout CYP2K1, rabbit anti-trout CYP2M1 (both gifts from Donald R. Buhler, PhD, Oregon State University, Corvallis, OR). Proteins of interest were visualized with goat anti-rabbit alkaline phosphatase conjugate (Bio-Rad) and the Western Breeze Novex Western Blot Immunodetection Kit (Invitrogen) according to the manufacturer's directions using Western Lightening CDP-Star Chemiluminescent reagent (Perkin Elmer Life Sciences, Boston, MA). Image analysis was accomplished using the VersaDoc 3000 Imaging System and QuantityOne analysis software (Bio-Rad). A summary of CYP1A results of the pilot study samples was previously presented (Codi King et al., 2005a) in which the samples were analyzed with a different primary antibody (anti-trout CYP1A1 277-294/KLH ) under different SDS-PAGE conditions (mini-Protean II, individually poured gels in the previous study) and with different methods for quantification (film, scanning densitometry and NIH Image ® in the previous study). For this study, a commercially available antibody was used and the number of samples that could be compared on one gel was increased improving the statistical analyses.

Densitometry and Statistical Analysis

The optical density of the samples is given as a Trace Quantity (QTY; Bio-Rad QuantityOne) or the quantity of a band as measured by the area under its intensity profile curve. Trace QTY units are intensity × area. For standardization, one sample in triplicate was loaded on each gel. The mean Trace QTY of this sample was used as a gel factor for standardization between gels. Trace QTY results are presented as an Arbitrary Optical Density (without gray-scale calibration). Statistical analysis was performed using GraphPad Prism . For the pilot study, a two-way ANOVA was performed using both gender and site as covariates. The results indicated that for each individual isozyme site was a significant factor, but not gender, and there were no interactions between these two factors. Therefore, for the one-way ANOVA the males and females were pooled, and the ANOVA results were analyzed by Turkey's multiple comparison test (P < 0.05, 0.01 and 0.001). Similar analysis of covariates allowed the use of the same statistical approach for the caged fish study.

Results

Pilot study

The results of the immunodetection of the CYP1A-, CYP2M1-, and CYP2K1-like proteins indicated that proteins of the appropriate molecular weight range for each isozyme were detectable in Gold-Spotted Trevally. Most Gold-Spotted Trevally showed one immunodetectable band of CYP1A-, CYP2M1-and CYP2K1-like proteins; some samples had a second CYP2M1-like protein. Because this second protein was not present in each sample, only the first band of lower molecular weight was quantified (Figure 3). Similar to the previous study (Codi King et al., 2005a), CYP1A-like protein levels in Gold-Spotted Trevally from the near-field (Harriet A) site were significantly higher than those from the far-field (Harriet C) site and the reference site. Although not significant, Gold-Spotted Trevally also exhibited a trend of increased CYP2M1-like protein levels at Harriet A as compared to Harriet C and the reference site (Figure 3).

For CYP2K1-like protein, a significant increase was observed at the Harriet C site compared to the reference site (Figure 3). The induction of CYP2K1-like proteins at the Harriet C site in Trevally may be due to possible exposure to hydrocarbons from natural oil seepages found in this region, or due to the movement of fish between platforms. The elimination of these variables was one of the main reasons for undertaking the subsequent controlled field study using caged fish.

Caged fish study

Immunodetection of CYP1A-like proteins indicated that proteins of the appropriate molecular weight range were detectable in Stripey seaperch at all three sites and over the time of the exposure (Figure 4a). The results for the CYP1A-like proteins (Figure 4b) demonstrated that by Day 3 of exposure both Site A and Site B had higher mean values than Site C, albeit not significant. By Day 10, fish collected at Site A had significantly higher induction of CYP1A-like proteins as compared to the fish at Site B and Site C. Comparisons also demonstrated that Site A fish after 10 days of exposure had significantly induced levels of CYP1A-like proteins as compared to the holding tank fish (T=0).

Figure 5a presents the optical densities for the species-specific pattern of CYP2M1-like proteins determined in Stripey seaperch, with two distinct appropriate molecular weight proteins in most samples; CYP2M1-like Protein 1 (52.8 kDa) and CYP2M1-like Protein 2 (49.0 kDa). Immunodetection of CYP2M1-like proteins demonstrated a similar pattern of expression for both Protein 1 and Protein 2 in Stripey seaperch with significant induction occurring at Day 10 for both proteins (Figures 5b and5c) and at Day 3 the same increasing but not significant trend. For Site A both Day 3 and Day 10 exposed Stripey seaperch had significantly higher levels of CYP2M1-like proteins than the holding tank fish. It is interesting that Site C Day 3 exposed fish also demonstrated site-specific differences from the holding tank fish.

Figure 6a presents the optical densities for the species-specific pattern of CYP2K1-like proteins determined in Stripey seaperch, again showing two distinct appropriate molecular weight proteins; CYP2K1-like Protein 1 (52.5 kDa) and CYP2K1-like Protein 2 (48.2 kDa). Unlike the two CYP2M1-like proteins, the induction patterns for the two CYP2K1-like proteins were dissimilar. For CYP2K1-like Protein 1, levels (Figure 4b) were significantly elevated for Site A Day 3 as compared to fish collected on Day 3 from Site B and Site C. At Day 10 there were no site-specific differences in expression, although Site A Day 10 and Site A Day 3 Stripey seaperch had significantly higher CYP2K1-like Protein 1 levels than the holding tank fish. For CYP2K1-like Protein 2 levels (Figure 4c) the induction trend was similar to the pattern observed for CYP1A-and both CYP2M1-like Protein 1 and Protein 2 in that by Day 10, Site A fish demonstrated the highest mean level of induction. The same site-specific differences were also observed with Site A Day 10 fish and Day 3 fish having significantly higher CYP2K1-like Protein 2 induction levels than the holding tank fish.

Discussion

Fish are important organisms for monitoring environmental contaminants because of their position in the food web, their lifestyle, their relative abundance and their adaptable physiology. Fish CYP1A induction is an extremely useful biomarker of chemical exposure in the environment [START_REF] Flammarion | Multibiomarker Responses in Fish from the Moselle River (France)[END_REF], particularly for persistent pesticides [START_REF] Levine | Modulation of CYP1A expression in rainbow trout by a technical grade formulation of propiconazole[END_REF], polychlorinated biphenyls [START_REF] Burton | Circulating lysozyme and hepatic CYP1A activities during a chronic dietary exposure to tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) mixtures in channel catfish, Ictalurus punctatus[END_REF][START_REF] Haasch | Caged and wild fish: Induction of hepatic cytochrome P-450 (CYP1A1) as an environmental biomonitor[END_REF], dioxins [START_REF] Zodrow | Histological analysis of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish[END_REF][START_REF] Smith | Environmental polychlorinated biphenyl exposure and cytochromes P450 in raccoons (Procyon lotor)[END_REF], pulp mill effluents [START_REF] Oikari | Bioavailability to juvenile rainbow trout (Oncorhynchus mykiss) of retene and other mixed-function oxygenase-active compounds from sediments[END_REF], xenoestrogens [START_REF] Katchamart | Effect of xenoestrogen exposure on the expression of cytochrome P 450 isoforms in rainbow trout liver[END_REF], spilled crude oil [START_REF] Jewett | Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in nearshore demersal fishes[END_REF], and polycyclic aromatic hydrocarbons (PAHs) [START_REF] Stagg | Effects of polycyclic aromatic hydrocarbons on expression of CYP1A in salmon (Salmo salar) following experimental exposure and after the Braer oil spill[END_REF]. The induction of CYP1A has implications for human fish consumption, as well as for the health status of aquatic organisms [START_REF] Arinc | Cytochrome P4501A and associated mixed-function oxidase induction in fish as a biomarker for toxic carcinogenic pollutants in the aquatic environment[END_REF].

The induction of CYP2 family proteins in bluegill and catfish due to exposure to peroxisome proliferating agents (PPAs; [START_REF] Haasch | Induction of anti-trout lauric acid hydroxylase immunoreactive proteins by peroxisome proliferators in bluegill and catfish[END_REF] was the first description of an inducible P450 in fish other than CYP1A. The CYP2M1-and 2K1-like proteins in bluegill and catfish were later shown to be lauric acid hydroxylases [START_REF] Haasch | Induction of lauric acid hydroxylase activity in catfish and bluegill by peroxisome proliferating agents[END_REF]. Bivalves exposed to petroleum water-accommodated fraction (WAFs) produce peroxisome proliferation [START_REF] Cajaraville | Peroxisome proliferation in the digestive epithelium of mussels exposed to the water accommodated fraction of three oils[END_REF]. Therefore, petroleum hydrocarbon, as the major contaminant in PFW, may be considered a PPA, even though the mechanism is unknown. These findings provided an indication that a petroleum hydrocarbon-initiated peroxisome proliferation response may occur in lower vertebrates similar to the model PPA-mediated induction of CYP2M1/2K1 [START_REF] Ackers | Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus[END_REF][START_REF] Johnson | Role of the peroxisome proliferator-activated receptor in cytochrome P450 4A gene regulation[END_REF].

These two CYP2 family P450 isozymes have been most thoroughly characterized in rainbow trout, (Oncorhynchus mykiss; [START_REF] Buhler | Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring[END_REF]. The molecular weight of CYP2K1 in rainbow trout is about 54 kDa, and has been shown to catalyze the ( -1) and ( -2) hydroxylation of lauric acid and some longer chain fatty acids; the molecular weight of CYP2M1 in rainbow trout is about 50 kDa, and this isozyme is the only known P450 responsible for laurate hydroxylation at the ( -6) position. Both isozymes also catalyze the hydroxylation of estradiol, testosterone and progesterone [START_REF] Buhler | Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring[END_REF]. It follows that induction of CYP2K1-and CYP2M1-like proteins in fish may have effects on fatty acid and steroid homeostasis, with possible links to developmental and reproductive toxicity as well as carcinogenesis [START_REF] Biegel | Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies[END_REF][START_REF] Vanden | Perfluorodecanoic acid as a useful pharmacologic tool for the study of peroxisome proliferation[END_REF][START_REF] Vanden | Inhibition of long-chain acyl-CoA synthetase by the peroxisome proliferator perfluorodecanoic acid in rat hepatocytes[END_REF].

A variety of structurally diverse xenobiotics and pharmaceuticals are known to produce peroxisome proliferation and concordant fatty acid metabolizing enzyme induction in mammals, therefore, the monitoring of CYP2 family isozymes in fish may have implications for human health and has the potential to become an important indicator of environmental health.

The immunodetection results indicated one highly immunoreactive protein for CYP1A and multiple immunoreactive proteins for the CYP2-like proteins. Some cross-reactivity between CYP2-like proteins is expected with the polyclonal anti-CYP2K1 and anti-CYP2M1 antibodies (some affinity for the same protein epitopes) as these antibodies have cross-reactivity to both rainbow trout CYP2K1 and CYP2M1 [START_REF] Buhler | Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring[END_REF]. Further evidence for cross-reactivity was evident in samples that also included one or more minor proteins of intermediate size that were specific to individuals and not consistently represented across all individuals. The CYP2 family is known to be fairly complex in number of isozymes and expression levels [START_REF] Buhler | Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring[END_REF]. Multiple cross-reactivity was not unexpected and did not detract from the relatively clear induction of two proteins using the anti-CYP2 antibodies. The proteins were designated CYP2M1-and CYP2K1-like proteins because appropriate standards for the tropical fish species do not yet exist and were beyond the scope of this study. Nevertheless, it is evident that the CYP2 family antibodies recognize constitutively present and inducible proteins that could potentially be used as biomarkers. Furthermore, it is possible, with appropriate sequence information, for commercially-available anti-CYP2 family antibodies to be developed.

The pilot study CYP1A results supported the findings of an earlier analysis of those samples using a different methodology indicating induction of CYP1A (Codi King et al., 2005a). Unlike the previous analysis, the current study did not indicate gender-specific responses. Multiple differences between the two analyses could contribute to the discrepancy including the use of a different primary antibody preparation. Similar to the previous analysis, site-specific responses were detected.

Analysis of the pilot study samples for CYP2M1-and CYP2K1-like proteins indicated possible site-specific induction and prompted the further examination of these proteins in the caged fish study.

Studies of environmental contaminant-mediated induction of the cytochromes P450 in tropical fish are lacking (Codi King et al., 2005a;[START_REF] Vrolijk | Toxicological and ecological implications of biotransformation enzymes in the tropical teleost Chaetodon capistratus[END_REF]. Both the pilot and caged fish field studies indicated significant results pertaining to the induction of cytochromes P450 in tropical fish species by PFW. First, the induction of CYP1A in Gold-Spotted Trevally and Stripey seaperch at the near-field site indicated that even though PFW discharge limits are being met [START_REF] King | Chemical and biological assessment of produced formation water (PFW) effluent from Harriet A Platform, on a tropical reef species, Stripey searperch (Lutjanus carponotatus)[END_REF], exposure to CYP1A inducing chemicals within the diluted PFW produced a significant induction effect. Numerous studies have demonstrated that chronic low level exposure of fish and especially larvae to PAHs can have a significant impact and can produce long-term, adverse health and reproductive effects [START_REF] Incardona | Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons[END_REF][START_REF] Stegeman | Cytochrome P450 1A expression in midwater fishes: potential effects of chemical contaminants in remote oceanic zones[END_REF]. These adverse effects are most likely to be more serious to those organisms attached to the platform or living under or near the platform in a restricted range but could also be detrimental to organisms cyclically exposed to pools of PFW.

Second, the significant induction of the CYP2K1/2M1-like proteins in both the In support of the previous findings, the results indicate that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1-and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and CYP2 family induction may represent possible new biomarkers (other than CYP1A) associated with PFW. In addition, the novel response of one CYP2K-like protein requires further verification but offers promise for improved monitoring of sub-lethal responses in marine organisms.

  Gold-Spotted Trevally and the Stripey seaperch is the first documented significant environmental induction in fish of any cytochrome P450 isozyme other than CYP1A1. The similar induction pattern of CYP1A1 and CYP2K1-like Protein 2 and CYP2M1-like Protein 1 and 2 may indicate that all of these isozymes are induced by the same chemical class contained within the PFW. While this finding alone is a reason for further study, it is perhaps even more interesting that there is an apparent response difference for induction of particular CYP2K1/2M1-like proteins. Only the CYP2K1-like Protein 1 was induced at the near-field site (Site A) on Day 3, but not on Day 10. The apparently different induction of the CYP2K1 Protein 1 compared to the other CYP isozymes potentially represents a unique chance to develop another bioindicator of PFW exposure. Although the metabolic activities of the CYP2M1 and CYP2K1 isozymes in Stripey seaperch are not known, it is reasonable to assume that the fatty acid and reproductive steroid hormone substrates would differ among the different proteins. Induction of different isozymes may therefore have different shortand long-term effects on the exposed organisms.

Figure 1 :

 1 Figure 1: Pilot study site locations: Harriet A, Harriet B, Harriet C, and the reference sites, indicated by triangles, off the Montebello Islands.

Figure 2 .

 2 Figure 2. Site locations of the caged fish study: (Site A ~200 m, Site B ~1000 m and Site C ~20 km) relative to the Harriet A PFW discharge.

Figure 3 .

 3 Figure 3. Pilot study: the species specific pattern of CYP1A-, CYP2M1-, and CYP2K1-like proteins determined in Gold-Spotted Trevally (shown for comparison of isozyme expression not for illustration of site effects), and graphical representation of the optical densities (Mean ± SD) determined for each site. The molecular weight range of the proteins is typical of cytochromes P450 and is between 45 and 60 kDa for all three isozymes. Numbers above the bars indicate the number of female and male fish (e.g., 6/2, female/male) represented in the average densitometry values. No statistical difference was observed between genders.

Table 1 :

 1 Characteristics of Stripey seaperch used in the caged fish study. All of the 383 fish were of intermediate reproductive status (sex-stage) and had plasma vitellogenin 384 levels that were below the detection limit.

	385
	Holding Tank or	Time	Sex Ratio	Body Weight *	Body Length *	Age *
	Cage Site	(days)	(Female/Male)	(g)	(mm)	(yr)
	Holding Tank	0	(4/7)	271 + 85	261 + 22	5 +
	Reference Site C	3 10	(4/4) (4/4)	292 + 83 284 + 52	265 + 30 257+ 19	6 + 4 +
	Far-Field Site B	3 10	(0/5) (0/5)	273 + 123 324 + 92	260 + 34 269 + 21	5 + 3 +
	Near-Field Site A	3 10	(3/5) (1/4)	371 + 88 293 + 79	285 + 20 262 + 28	5 + 5 +

*Values are mean ± SD for male and female combined. 386

ACKNOWLEDGMENTS

This research was supported by the Environmental Toxicology Research Program of the University of Mississippi, and by the Australian Institute of Marine Science and Apache Energy Pty Ltd. We would like to thank the entire crew of the Lady Basten (pilot study) and the entire crew of the Cape Ferguson (caging study) for their assistance in the field. We would also like to thank Mary Johnston of the University of Maryland, Chesapeake Biological Laboratory for technical assistance on the Pilot Study samples, Cary McLean of AIMS for technical expertise during the caging study and Craig Skepper from WA Fisheries for all his assistance during the caging study.


Yana Yudkovski

Adelina Rogowska-Wrzesinska

Irena Yankelevich

Edna Shefer

Barak Herut

Moshe Tom
email: tom@ocean.org.il

Quantitative immunochemical evaluation of fish metallothionein upon exposure to cadmium

Keywords: Biomarker, metallothionein, beta-actin, Lithognathus mormyrus, Mediterranean, real time PCR, ELISA, cadmium, biomonitoring

Efficient implementation of an environmental biomarker requires multi-annual comparability over a wide geographical range. The present study improved the comparability of a quantitative competitive metallothionein (MT) Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) in the sentinel fish Lithognathus mormyrus by introducing to the assay recombinant MT and beta-actin standards.

Commercial antibodies for cod MT and mammalian actin were implemented. In addition, a sensitive anti L. mormyrus MT antibody was produced, adequate only for solid phase immunochemical assays. Cadmium was applied to the fish through injection and feeding to serve as a testing platform of the ELISA. The results demonstrated high potential protective capacity of the liver against toxic levels of transition metals through increasing MT levels. MT transcript levels were evaluated also from fish sampled at polluted and relatively clean natural sites, indicating applicability of MT as biomarker of exposure to a multi-factorial pollution, in comparison to its low revealed sensitivity to controlled cadmium exposure.

Introduction

Levels of environment-affected transcripts and proteins, evaluated in a native sentinel species are widely used as environmental biomarkers of an examined habitat. The appropriate establishment of diagnostic environmental gene-product biomarkers in general and in fish was recently discussed in [START_REF] Handy | A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology[END_REF], [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF] and [START_REF] Tom | Transcript and protein environmental biomarkers in fish -a review[END_REF]. The latter review defined long-term comparability of biomarker levels over a wide geographical range as an essential requirement from an environmental biomarker. Evaluations done by more than one laboratory would further complicate the comparisons. The suggested components of an environmental bio monitoring and its characteristics include: (1) standard proteins or transcripts, enabling absolute quantification of the biomarker levels. Recombinant products are preferred, securing supply and repeatability of the standards. (2) Normalization using constitutively expressed gene products which are processed concurrently with the target biomarker and evaluated by similar methods. (3) The range of the sub-lethal biomarker values have to be determined experimentally, scaling the levels obtained from feral sentinels within the attainable range.

A series of studies performed in our laboratory [START_REF] Tom | Competitive ELISA utilizing membrane-free recombinant fish CYP1A as standard for evaluation of its microsomal homologue molar concentrations[END_REF], 2003, 2004[START_REF] Funkenstein | Application of real time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped seabream, Lithognathus mormyrus[END_REF] used the above guidelines to adapt two generic methods to be implemented as environmental biomarker assays, using the striped sea bream (Lithognathus mormyrus) as sentinel fish.

Quantitative real time PCR was used for transcripts evaluations and Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) for proteins. Both methods were modified to include appropriate specific transcript or protein standards compatible with the target fish. Specific PCR primers and antiprotein antibodies were used as detection probes.

The striped sea bream was selected as sentinel fish of its native sandy coastal habitat as it is widely distributed throughout the entire Mediterranean and also in the South-Eastern Atlantic [START_REF] Bauchot | Sparidae[END_REF]. It is a bottom-dwelling fish, inhabits polluted and pristine coastal sandy habitats and interacts with the water column but also with the bottom, through its bottomfeeding habits and burial behavior [START_REF] Suau | Contribucion al estudio de la biologia de Lithognathus (Pagellus) Mormyrus L. (Peces esparidos)[END_REF]. Its gut usually contains sediment, engulfed with the ingested food [START_REF] Froglia | Feeding of Lithognathus mormyrus (L.) in the Central Adriatic Sea (Pisces, Sparidae). Rapport et Procès-Verbaux des Réunion Commission Internationale pour L' exploration[END_REF][START_REF] Buxton | Inshore small-mesh trawling survey of the Cape south coast. Part 4. Contributions to the biology of some Teleostei and Chondrichthyes[END_REF][START_REF] Lasiak | Aspects of the biology of three benthic-feeding teleosts from Kingmins Beach, Algoa Bay[END_REF]. It is a protandrous hermaphrodite [START_REF] Besseau | Etude Histo-Cytologique de la structure sexuelle d`une population de Lithognathus mormyrus (L.) (Teleosteen, Sparide)[END_REF][START_REF] Kraljevic | Sexual maturation, age and growth of striped sea bream, Lithognathus mormyrus L., on the eastern coast of the Adriatic Sea[END_REF], similar to other members of its family, the Sparidae [START_REF] Buxton | Alternative reproduction styles in seabreams (Pisces, Sparidae)[END_REF][START_REF] Micale | Further studies on the sexuality of the hermaphroditic teleost[END_REF]. The gonad is an ovotestis, sexually indistinguishable outside the summer reproductive season. In addition, there are also no external sex-distinguishing characters [START_REF] Bauchot | Sparidae[END_REF][START_REF] Kraljevic | Sexual maturation, age and growth of striped sea bream, Lithognathus mormyrus L., on the eastern coast of the Adriatic Sea[END_REF].

Metallothionein (MT) is a 6-7 kDa metallo-sulphur rich protein [START_REF] Hamer | Metallothionein[END_REF][START_REF] Kagi | Overview of metallothionein[END_REF].

Hepatic MT synthesis is induced by a number of metals, cytokines and stress hormones as well as by a wide range of chemicals, many of which act indirectly via a stress or inflammatory response [START_REF] Coyle | Metallothionein: the multipurpose protein[END_REF]. Its transcript and protein are induced in fish by a variety of bi-valent metals, including cadmium, zinc, copper, lead and mercury and were widely used as biomarkers for evaluating the cumulative biological effect of transition metals disposed to the marine environment [START_REF] Chan | Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues[END_REF][START_REF] George | Metallothioneins as indicators of trace metal pollution[END_REF][START_REF] George | Regulation of metallothionein in teleosts: Induction of metallothionein mRNA and protein by cadmium in hepatic and extrahepatic tissues of a marine flatfish, the turbot (Scophthalmus maximus)[END_REF][START_REF] Olsvik | Brown trout (Salmo trutta) metallothioneins as biomarkers for metal exposure in two Norwegian rivers[END_REF][START_REF] Tom | Monitoring of hepatic metallothionein mRNA levels in the fish Lithognathus mormyrus -evaluation of transition metal pollution in a Mediterranean coast[END_REF][START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilisation as an environmental biomarker[END_REF][START_REF] Atli | Natural Occurrence of Metallothionein-Like Proteins in the Liver of Fish Oreochromis niloticus and Effects of Cadmium, Lead, Copper, Zinc, and Iron Exposures on Their Profiles[END_REF][START_REF] De Boeck | Differential metallothionein induction patterns in three freshwater fish during sublethal copper exposure[END_REF][START_REF] Eroglu | Effects of metal (Cd, Cu, Zn) interactions on the profiles of metallothionein-like proteins in the nile fish Oreochromis niloticus[END_REF][START_REF] Long | Metallothionein induction and bioaccumulation kinetics of Cd and Ag in the marine fish Terapon jarbua challenged with dietary or waterborne Ag and Cu[END_REF][START_REF] Woo | Heavy metal-induced differential gene expression of metallothionein in Javanese medaka, Oryzias javanicus[END_REF][START_REF] Alvarado | Quantitative changes in metallothionein expression in target cell-types in the gills of turbot (Scophthalmus maximus) exposed to Cd, Cu, Zn and after a depuration treatment[END_REF][START_REF] Hansen | Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels[END_REF].

Transition metals are found in the marine environment as free ions, as well as in a variety of complexes with suspended particles and sediments. Fish are exposed to the various transition metal species at different intensities. There are two major routes of metals exposure. Metal ions dissolved in the ambient water are absorbed through the gills [START_REF] Alvarado | Quantitative changes in metallothionein expression in target cell-types in the gills of turbot (Scophthalmus maximus) exposed to Cd, Cu, Zn and after a depuration treatment[END_REF] and other permeable body surfaces. Metals bound to solid particles are ingested, detached from their carrier particles in the digestive system and absorbed through the gut epithelium [START_REF] Berntssen | Tissue metallothionein, apoptosis and cell proliferation responses in Atlantic salmon (Salmo salar L.) parr fed elevated dietary cadmium[END_REF].

Four parameters were basically used to evaluate MT protein levels: MT metal content, metal substitution, the amount of MT sulfhydryl groups and MT immunochemical affinity [START_REF] Hylland | Biological effects of contaminants: quantification of metallothionein in fish[END_REF]. A competitive ELISA was described by Hylland (1999) using a native purified cod MT as standard protein and anti-cod MT antibody. [START_REF] Wu | Cadmium-Inducible Metallothionein in Tilapia (Oreochromis mossambicus)[END_REF][START_REF] Wu | Metallothionein Induction in Early Larval Stages of Tilapia (Oreochromis mossambicus)[END_REF] described similar ELISA for tilapia using partial synthetic MT as coating buffer and the native purified protein as standard. The major aim of the present study was to adapt this competitive ELISAs to L. mormyrus and its improvement by introducing recombinant MT as standard. Parallel ELISA of beta-actin was developed to be used as normalizing agent, applying recombinant beta-actin as standard. A byproduct of this study was the production of sensitive anti L. mormyrus MT antibody. Unfortunately, this antibody reacted only with MT immobilized on solid phase, therefore, was not adequate for our competitive ELISA.

In vitro production of recombinant MT in E. coli was carried out in both mammals and fish [START_REF] Vergani | Expression, purification, and characterization of MT-A from rainbow trout[END_REF][START_REF] Yang | High-yield expression in Escherichia coli of soluble human MT2A with native functions[END_REF]. eta-actin was in vitro produced in mammals using Sf9 insect cell host and baculovirus vector [START_REF] Joel | Expression of a nonpolymerizable actin mutant in Sf9 cells[END_REF].

Exposure of L. mormyrus to food-borne and injected cadmium was served as platform for testing the two developed ELISAs. It was used also to simultaneously measure hepatic levels of MT transcript and protein as well as the corresponding levels of bio-accumulated cadmium in relation to the applied metal ions. In addition, hepatic MT transcript levels were measured in feral fish sampled at two natural habitats of L. mormyrus and served to compare realistic environmental situation to controlled exposure to a single pollutant.

Material and Methods

General molecular methods

General biochemical and molecular protocols followed [START_REF] Sambrook | Molecular cloning, a laboratory manual[END_REF] and include RNA electrophoresis, spectrophotometric evaluation of RNA concentrations, plasmid manipulations and selective growth of bacteria on Luria-Bertani (LB) medium-agar plates. PCR assays, excluding real time PCR, were done in final concentrations of 0.05 units l -1 Taq DNA polymerase, in buffer provided with the enzyme. The solution also contained 0.2 mM of each dnucleotide triphosphate, and primer concentrations of 10 M. PCR designs and applied templates are detailed below for each specific reaction.

Synthesis of recombinant MT

The synthesis generally followed [START_REF] Vergani | Expression, purification, and characterization of MT-A from rainbow trout[END_REF] with several modifications. The coding region of L. mormyrus MT (GenBank Accession no. AF321007) was modified by PCR (95°C -2 min; 35 cycles of [95°C -1 min, 51°C -1 min, 72°C -1 min]; 72°C -2 min) to include EcoRI and XhoI restriction sites at its two ends. The MT cDNA-containing vector served as PCR template, applying the primer pair 5'-TCCCCGGAAT TCATGGACCC GTGCGAGTGC-3' and 5'-ATGGGGCCGC TCGAGTCACT GACAGCAGCT AGTG-3'. A glutathione S-transferase (GST)-MT fusion protein was formed in pGEX-4T-1 vector (Amersham, UK) by ligating the MT PCR product to the vector through the two restriction sites. The recombinant vector was transfected into competent BL21-Gold (3D) bacterial strain (Stratagene, San Diego, USA). Selected BL21 colony was grown overnight at 37ºC with shaking (~250 rpm). Isopropyl β-D-thiogalactopyranoside (IPTG) and Zn ++ were added at mid-exponential bacterial growth phase (OD 600 ~ 0.6) to final concentrations of 0.1 mM and 300 µM, respectively. Induced cells were grown for additional four hours, harvested by centrifugation, and washed in PBS (8.1 mM Na 2 HPO4, 1.47 mM KH 2 PO4, 137 mM NaCl, and 2.7 mM KCl; pH 7.3) containing 5 mM dithiothreitol (DTT). Bacterial cells were lysed by their resuspension in PBS (4 ml/100 ml culture) containing 5 mM DTT and supplemented with protease inhibitors cocktail and 1 mg ml -1 lysozyme, mildly rocked for 30 min at 4°C. 2.5 volumes of 0.2% triton X-100 (in PBS) was injected forcibly into the viscous cell lysate concurrent with 1 µg ml -1 DNase and the lysate was rocked for additional 10-20 min. The lysate was centrifuged at 10,000 g for 30 min, and the supernatant was recovered and supplemented with 1 mM DTT. The GST-MT fusion protein was purified by batch affinity chromatography with glutathione-Sepharose-4B beads (Pharmacia, Uppsala, Sweden) at a volume ratio of 1/10 (matrix/sample). The mixture was incubated with gentle agitation for 30-60 min at room temperature, and after washing (×3) in PBS, the resin was packed in a column and the MT-GST was eluted with glutation/Tris buffer (10 mM reduced glutathione in 50 mM Tris-HCl; pH 8). MT-GST containing fractions were 10-fold concentrated using Centriprep concentrators (Amicon, Billerica, USA; cutoff of 3 kDa) and the MT was cleaved from the GST in thrombine-PBS solution (10 units thrombine mg -1 fusion protein, Pharmacia) for 16h at 22°C. To that end, the solution was heated for 30 min at 75-80ºC, followed by centrifugation at 20,800 g for 20 min.

L. mormyrus cytosolic fraction was separated from hepatic homogenates in PBS by ultracentrifugation according to [START_REF] Tom | Competitive ELISA utilizing membrane-free recombinant fish CYP1A as standard for evaluation of its microsomal homologue molar concentrations[END_REF] and the resulting supernatant was heated to 70°C for 20 min. The denatured proteins were precipitated at 20,800 g for 20 min and the resulting supernatant was concentrated as required for visualization of the MT by Western blot.

An anti-MT antibody was produced in rabbits using the recombinant protein as antigen but did not immunoreact with the native protein in the ELISA solution, therefore, replaced by a much less sensitive commercial polyclonal anti-Cod IgG (Abcam, Cambridge, UK; #ab36882).

Mass spectrometry sequencing of the recombinant MT Recombinant MT (1 mg) in PBS was freeze-dried and dissolved in 20 µl of 8 M urea and 0.4 M NH 4 HCO 3 buffer. Subsequently, 20 µl of EDTA and 5 µl of 45 mM DTT were added and the sample was incubated at 50°C for 15 min. After cooling down to room temperature, 5 µl of 100 mM of iodoacetamide was added and the solution was incubated for 30 min at room temperature in the dark. The reduced and alkylated protein was digested with 2% trypsin (Promega, Madison, WI, USA,) for 24 h and the peptide mixture was separated using high performance liquid chromatography on AKTAexplorer TM system (GE Healthcare, UK). The fractions were analyzed by Matrix Assisted Laser Desorption-Time of Flight-tandem mass spectrometry (MADLI-TOF MS/MS) using the 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA).

Synthesis of recombinant beta-actin

The coding region of L. mormyrus beta-actin cDNA was reverse transcribed. The resulting cDNA was modified to contain the PstI and EcoRI restriction sites, as well as a 5×histidine tag at its carboxy terminus. The modification was carried out by PCR (95°C-2 min; 35 cycles of [95°C-1 min, 55°C-1 min, 72°C-3 min]; 72°C-2 min) using the primer pair 5'-ATGGAAGATG AAATCGCCGC AC-3' (forward/PstI) and 5'-CTAATGGTGATGG TGATGGAAGC ATTTGCGGTG GA CG-3' (reverse/EcoRI; 5×histidine tag in bold italics). The primers were designed according to the beta-actin sequence of the related sparid fish Sparus aurata (GenBank Accession no. X89920). Beta-actin was produced in Sf9 insect cells according to the instructions of the production kit manufacturer (Pharmingen, San Diego, CA, USA) excluding the Sf9 cell lysis procedure. Briefly, the modified cDNA was cloned into the pVL1392 baculovirus transfer vector and than co-transfected with the Baculogold viral DNA into Sf9 insect cells, maintained in monolayer culture. Cells were harvested 72h post-infection by low speed centrifugation at 100 g for 5 min, and lysed for 30 min at 4°C in insect cell lysis buffer (0.5 ml/100 mm plate) composed of 50 mM sodium phosphate buffer (pH 7.4), 450 mM NaCl, 1% Triton X-100 and 10% glycerol. The [START_REF] Herut | Trace metals in shallow sediments from the Mediterranean coastal region of Israel[END_REF]2006). Fish were maintained at the laboratory, before and during the experiment, in 0.7 m 3 tanks supplied with flow-through Mediterranean coastal seawater at ambient winter seawater temperature of 16-21°C, fed by commercial fish food pellets (Cat. # 1369, Matmor central feed mill, Ashquelon, Israel).

Cadmium-containing food was prepared by grinding the food pellets and their repackaging as a mixture of 63% food powder, 35% CdCl 2 aqueous solution (200 or 20 µg CdCl 2 ml -1 ) and 2.5% gelatin as solidifier, dried overnight at 45-50°C. Cadmium was applied also through 50 µl intra peritoneal injections of CdCl 2 solution in saline (2 mg cadmium ml -1 ).

Fish were decapitated at the termination of each experiment or immediately after sampling, followed by an immediate dissection of liver and gonads. Livers were divided into smaller aliquots, snap frozen in liquid nitrogen and kept at -80°C. Gonads were fixated in 4% formaldehyde solution. Fish reproductive activity was determined by microscopic observation of gonads smeared between two slides, measuring the average ova diameter of 20 oocytes. Being proteandrous hemaphrodite, L. mormyrus ovotestes contained always at least primordial oogonia.

MT induction experiment

Table 1 provides detailed description of each experimental group of fish, including treatment, total number of fish, average fish weight, average oocyte diameter, and description of the treatment.

Protein ELISA evaluations were carried out on protein pools, as the futile examinations of the inhouse produced antibody (see above) left us with too small protein samples. The number of evaluated protein samples is also detailed in table 1.

MT, beta-actin and cytochrome P4501A competitive ELISAs Fish hepatic homogenates, 500 µl/60-80 mg tissue, were prepared in PBS, supplemented prior to use, with 5 mM beta-Mercaptoethanol and protease inhibitors cocktail. Debris was eliminated by centrifugation at 10,000 g for 15 min, saving the supernatant. Purified recombinant cytochrome P4501A, MT and beta-actin were used as standards and their concentrations were determined by the modified Lowry method (DC protein assay kit, BioRad, Hercules, CA, USA). The competitive ELISAs were carried out according to [START_REF] Tom | Competitive ELISA utilizing membrane-free recombinant fish CYP1A as standard for evaluation of its microsomal homologue molar concentrations[END_REF] which described the ELISA of cytochrome P4501A. Triton X-100 application at the competition stage was implemented only for the ELISA of cytochrome P4501A.

A matrix of several concentrations of coating MT (100, 300, 500 nM) and antibody dilutions (1:2,000; 1:3,500; 1:5,000) were applied to optimize the ELISA. Similarly, coating beta-actin concentrations of 10, 50 nM and antibody dilutions of 1:10,000 and 1:20,000 were applied to optimize the corresponding ELISA. The optimization procedure was aimed at achieving wide dynamic range. The detailed optimization procedure is described in [START_REF] Tom | Competitive ELISA utilizing membrane-free recombinant fish CYP1A as standard for evaluation of its microsomal homologue molar concentrations[END_REF].

Serial dilutions of the standard protein and the samples were performed for each ELISA. Both the standard and the sample log-linear curves were used to identify the linear range of the immunochemical competition reaction, which was used for the analysis. Similarity of the curves' slopes between the sample-containing and the standard-containing ELISA wells signified similar immunochemical competition kinetics, an accuracy determining factor.

Evaluation of hepatic MT transcript levels

Total hepatic RNA was extracted using the EZ II kit (Biological Industries, Beit Haemek, Israel) or by the RNeasy purification kit (Qiagen), according to the manufacturers' instructions. The quality of all produced RNAs was evaluated by electrophoresis on a 1% agarose-formaldehyde gel and their concentration was evaluated by spectrophotometry.

MT and beta-actin transcript levels were evaluated by real time PCR according to [START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilisation as an environmental biomarker[END_REF] with a modification. The RNA templates for the reverse transcription reactions prior to the real time PCR were brought to a concentration of 0.5 ng µl -1 and a volume of 2 µl was applied into each reaction tube. Two µl of the resulted reverse transcription solution was added to each real time PCR reaction. The increased volumes in comparison to [START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilisation as an environmental biomarker[END_REF] and the equalized RNA levels were aimed at decreasing the variation among replicates.

Measurements of cadmium levels in fish liver

Cadmium concentrations were determined on approximately 0.1 g of dry liver tissue that was digested for 4 h at 140°C with concentrated nitric acid (65 wt %) in Uniseal, teflon-lined, high pressure decomposition vessels according to [START_REF] Herut | Particulate Metals Contamination in the Kishon River Estuary, Israel[END_REF]. The digests were diluted to exactly 15 ml in clean plastic tubes with double distilled water. Measurements were performed by Varian SpectrAA220 flame atomic absorption and SpectrAA880 graphite furnace. The accuracy and precision of the methods were evaluated on the basis of analysis of several international standard reference materials (DOLT3, DORM-2, IAEA 407). All elements gave results within 5% of the certified values.

Results

Production of recombinant MT

The production and the purification procedure of the recombinant MT are demonstrated in Fig. 1, as well as the immunochemical affinity of both the anti-recombinant L. mormyrus MT and the commercial anti-cod MT to the recombinant protein and to hepatic extract of L. mormyrus. The isolated MT was identified as the major end product resulting from the purification procedure. The identity of the purified protein as MT was further demonstrated by mass spectrometry. Full sequences of 4 peptides (GSPEFMDPCECAK, TGTCNCGGSCSCTNCSCTSCK, KSCCSCCPAGCSK, SCCSCCPAGCSKCASGCVCK) were elucidated, with the third and the fourth peptides partially overlapping. An overall identity to 84% of the recombinant protein sequence was obtained. Peptides corresponding to the last 10 amino acids (GKTCDTSCCQ) were not recovered being fragmented into small peptides not detectable by the MALDI TOF-MS even when using an incomplete digestion approach. The first five amino acids of the first peptide (GSPEF) belong to the thrombin restriction site introduced by the pGEX-4T-1 vector.

Although protected from oxidation throughout the purification procedure, the recombinant MT revealed the typical ladder-like profile on SDS-PAGE, unlike the dimer conformation of the native MT. The antibody that was produced by us in rabbits using the recombinant protein as antigen was more sensitive than the commercial anti-cod MT when tested on solid surface. However, it had no affinity to the native MT in solution.

Production of recombinant beta-actin

The pure recombinant beta-actin revealed the appropriate actin molecular weight. Its identity was further demonstrated by its positive and specific immunochemical affinity to the commercial anti-actin which reacted also with the fish native protein. The resulting products of the various stages of the purification procedure and their immunochemical affinity to the antibody are presented in Fig. 2.

MT and beta-actin competitive ELISAs

Typical ELISA results of the two proteins, including the final optimized standard protein concentrations are presented in Fig. 3a (MT) and Fig. 3b (beta-actin). The optimal antibody dilutions were 1:5,000 (MT) and 1:20,000 (beta-actin). Optimal coated MT concentration was 300 nM ml -1 and the optimal coated beta-actin concentration was 10 nM ml -1 . Only the linear part of the ELISA reaction was used for the analysis of protein levels. It has to be emphasized that plate to plate variations in the binding capacity of the walls of the ELISA wells to the coated antigen may change the slope and the linear range of the competition reaction curve and the linear range has to be identified for each ELISA plate.

MT induction experiment

The results of the MT induction experiment are presented in Fig. 4. Levels of hepatic cadmium were normalized to liver wet weight, whereas MT transcript and protein were normalized to the respective levels of beta-actin transcript and protein.

Sampling of fish from natural habitats

The hepatic levels of MT and cytochrome P4501A, as well as cytochrome P4501A protein level in the sampled fish were evaluated. The results are detailed in Table 2. A statistically significant difference (t-test; p<0.05) was found between the two sampled sites for all the measured biomarkers, always higher in Haifa Bay. The gonads of all sampled fish were in reproduction arrest, revealing average sample oocyte diameters of: 83±30, 69±42, 53±26 and 61±32 µm, all below the size of reproductive females [START_REF] Funkenstein | Application of real time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped seabream, Lithognathus mormyrus[END_REF].

Discussion

The cadmium exposure experiment was aimed at creating several exposure intensities for testing the ELISAs. However, limited discussion of cadmium transportation to the liver and its sequestration by MT can be done in view of the results. Hepatic bio-accumulated cadmium levels corresponded to the applied cadmium levels for both types of treatment. The higher and lower ingested cadmium doses were 17.7-fold apart and the corresponding cadmium levels differed by 15.1-fold. The injected doses and the corresponding hepatic cadmium levels were both 4-fold apart.

Therefore, it can be postulated that cadmium rapidly reaches the liver, and during the experimental time scale was accumulated there. However, absorption of cadmium in the liver was much more efficient through the pharmacological injection route (17-18%) than through the physiological ingestion route (0.32-0.44%). Similar continuous cadmium accumulation was found in other fish cadmium exposure studies during a time frame of up to four months of exposure [START_REF] Berntssen | Tissue metallothionein, apoptosis and cell proliferation responses in Atlantic salmon (Salmo salar L.) parr fed elevated dietary cadmium[END_REF][START_REF] Chowdhury | Tissue-specific cadmium and MT levels in rainbow trout chronically acclimated to waterborne or dietary cadmium[END_REF]. The accumulated cadmium amounts in this study were very high due to the extreme exposure, indicating very high capacity of the liver for cadmium. Cadmium is accumulated in the liver and its further transportation to other organs is limited [START_REF] Wicklund | Cadmium and zinc interactions in fish: effects of zinc on the uptake, organ distribution, and elimination of 109 Cd in the zebrafish Brachydanio rerio[END_REF]. It seems that a chronic exposure which may lead to steady state cadmium hepatic levels requires long-term exposure to high cadmium levels and most exposure experiments, including ours, did not reach this equilibrium.

The results may be interpreted in view of [START_REF] Campbell | Cadmium-handling strategies in two chronically exposed indigenous freshwater organisms -the yellow perch (Perca flavescens) and the floater mollusc (Pyganodon grandis)[END_REF] who studied cadmium distribution in perch liver under several natural long-term, chronic exposure regimes to waterborne cadmium. They showed that fish hepatic cellular components, fractionated by differential centrifugation, sequestered cadmium in constant ratios. Cadmium was trapped by both cadmiumsensitive and resistant cellular components and the bio-accumulated amounts were in correlation with the exposure intensity. However, most of the cadmium (~60%) was located in the heat stable cytosolic component, probably trapped by MT, protecting the liver from cadmium toxicity. In the present study, the two cadmium-fed doses caused a 5-6-fold increase of both the MT transcript and protein levels. The cadmium application through feeding can be considered closer to equilibriumstate as determined by [START_REF] Campbell | Cadmium-handling strategies in two chronically exposed indigenous freshwater organisms -the yellow perch (Perca flavescens) and the floater mollusc (Pyganodon grandis)[END_REF] than the injected cadmium, as it was continuously provided to the fish over a two months period. The increased cadmium levels were sequestered by the increasing MT levels. One MT molecule can sequester 6-7 cadmium molecules [START_REF] Hamer | Metallothionein[END_REF].

Therefore, a 5-6 fold MT increase explained the trapping of 15-fold increased cadmium levels. In contrast, injected cadmium represent a pharmacological non-equilibrium state. Cadmium quickly entered the liver but the transcript and protein levels were still changing. Naturally, elevated MT transcript level, preceded the increase in protein levels, and the different durations of cadmium exposure between the two applied doses, 8 and 12 days for the low and high doses, respectively (Table 1) may explain the disproportional increase of the MT protein upon the high injection dose.

This extremely high MT level demonstrates high protection capacity of the liver from cadmium toxicity.

MT transcript level in the striped sea bream liver is used by us as an environmental biomarker, responding to the presence of environmental transition metals and other MT-affecting parameters.

However, exposure experiments to cadmium revealed MT transcript induction only upon exposure to relatively high levels of cadmium, not usually existing in the coastal environment of the Mediterranean. In this study, significantly induced MT transcript was related to a minimum of 7.4 µg cadmium/g wet liver weight, 10-20 fold higher than elucidated levels of cadmium and other MT inducing metals in the polluted Haifa Bay. Similar insensitivity was revealed by [START_REF] Bourdineaud | Challenging the model for induction of MT gene expression[END_REF] applying environmentally-realistic low waterborne cadmium to zebrafish, revealing no MT induction and [START_REF] Williams | Development of the GENIPOL European flounder (Platichthys flesus) microarray and determination of temporal transcriptional responses to cadmium at low dose[END_REF] who applied low cadmium levels to flounders with only faint and transient induction. A cadmium level of 0.49 µg cadmium/g wet liver weight, which was elucidated in one of the treatment groups, is much closer to real environmental levels, and it revealed no significant inducible MT transcript levels. However, the comparison of MT transcript levels between two sampling sites along the Israeli coast, revealed consistent differences in the levels of several biomarkers, including MT transcript, between polluted and clean sites over two years sampling period. Therefore, it can be preliminarily concluded that the chronic environmental influence over extended periods of time, composed of multiple effectors, evoked significant MT and also cytochrome P4501A induction, the latter emphasizes the multi-factorial nature of the pollution in the examined habitat. The partially undefined natural pollutant mixture is composed of cadmium and other transition metals [START_REF] Herut | Trace metals in shallow sediments from the Mediterranean coastal region of Israel[END_REF][START_REF] Herut | Environmental quality of Israel' s Mediterranean coastal waters in 2005[END_REF] and also other unknown components. Acute intensive cadmium exposures are interesting as they test the extremes of endurance and protection capacity. However, they do not mimic realistic environmental conditions which involve long-term exposures to low levels of multi-pollutant mixtures.

Recombinant MT and beta-actin standard proteins of the striped sea bream and suitable anti-MT and anti-actin antibodies were integrated into two competitive ELISAs to fit the striped sea bream MT protein evaluation and to improve its comparability. The beta-actin ELISA can also be used as a normalizing agent for the previously developed striped sea bream competitive ELISAs of cytochrome P4501A [START_REF] Tom | Competitive ELISA utilizing membrane-free recombinant fish CYP1A as standard for evaluation of its microsomal homologue molar concentrations[END_REF] and vitellogenin (methods are reviewed by [START_REF] Tom | Transcript and protein environmental biomarkers in fish -a review[END_REF]. The striped sea bream protein and transcript biomarkers, described here and in [START_REF] Tom | Quantitative evaluation of hepatic cytochrome transcript, protein and catalytic activity in the striped sea bream, Lithognathus mormyrus[END_REF][START_REF] Tom | Quantifying fish metallothionein transcript by real time PCR for its utilisation as an environmental biomarker[END_REF] and in [START_REF] Funkenstein | Application of real time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped seabream, Lithognathus mormyrus[END_REF] contain the components required for their implementation as comparable diagnostic environmental biomarker assays as depicted in the introduction above. However, establishment of these ELISAs as routine environmental assays requires further attention to two aspects: accurate and uniform protocol for standard protein evaluations, and improvement of the normalization procedure. The modified Lowry method used here for protein standard evaluation is not sufficiently accurate and the utilized protein standards used with the Lowry method (bovine serum albumin, gama-globulin, etc.) and their sources are not widely accepted and are subject to the decision of each laboratory.

Proper normalization agent should be a protein which is found in constant amounts per cell or tissue mass at all biological situations and also located in the same homogenized fraction as the target protein, and therefore exposed to the same homogenization procedure with similar extraction efficiency. It is difficult to find one universal protein to serve as a normalizing agent. Therefore, several agents have to be examined for every biomarker assay, selecting those which show better correlation with each other, statistically eliminating non-adequate ones. One protein, beta-actin, was provided by this study. It is a widely used normalizing agent and being present in the supernatant of the S9 fraction, it is adequate as MT, cytochrome P4501A and vitellogenin normalizing agent. The above discussion of measurement of standards and proper normalization is valid also to transcript evaluations.

Conclusions

Competitive ELISA for the measurement of MT levels was modified to be used in the sentinel fish L. mormyrus, alongside with a similar procedure for evaluating beta-actin levels to be used as a normalizing agent of MT and other evaluated biomarker proteins. Almost all the components of these ELISA procedures were described earlier in fish, excluding the production of recombinant fish beta-actin. However, their assembly into one environmentally-appropriate protocol may be considered the major contribution of this study. Two aspects of this biomarker evaluation system still require improvement: 1) accurate evaluation of the standards and 2) provision of additional normalizing agents for the selection of the best one for this assay. Both the transcript and the protein can serve as environmental biomarkers. It was demonstrated that although intensive controlled exposure to cadmium is required to induce MT transcript levels, the levels of MT in feral fish were significantly and repeatedly different between a polluted and clean natural habitats, although the accumulated level of metals in the sediment was lower than the level of the experimentally applied cadmium.

  lysis buffer was supplemented prior to use with 5 mM beta-Mercaptoethanol and protease inhibitors cocktail. Cellular debris was removed from the lysate by centrifugation at 20,800 g for 20 min. Recombinant Beta-actin was purified from the supernatant by affinity chromatography on a Ni-NTA column (Qiagen, Hilden, Germany) which was eluted from the column by a buffer which contained 200 mM imidazole. Both the recombinant and the native fish beta-actin reacted specifically with a commercial rabbit anti-actin IgG (Santa Cruz, Santa Cruz, CA, USA; #sc-1616r ,200 µg ml -1 ), immunoreacting with conserved actin C-terminus. Fish sampling, maintenance and experimental manipulations Fish were sampled alive in the winter from their natural habitats at the Mediterranean coast of Israel by gillnetting. A total of 44 striped sea bream individuals, sampled at the same date and site were used for the induction experiment. Another 112 fish were sampled at two coastal sites in the winters of 2004-2005 and were sacrificed three hours after sampling. The sediment and the biota of the Haifa Bay site are located adjacent to an industrial region and the Haifa Harbor and contain higher levels of transition metals than other sites along the Israeli coast of the Mediterranean. The second site consists of two locations 10-25 km south of Haifa bay that were considered clean sites

Fig. 1 :

 1 Fig.1: L. mormyrus MT was expressed in E. coli as MT-GST fusion protein, using the pGEX-4T-1

Fig. 2 :

 2 Fig.2: Recombinant 5×his tagged beta-actin was produced in Sf9 insect cells. Lane 1 -fish liver

Fig. 3 :

 3 Fig. 3: Examples of the results of MT and beta-actin ELISAs. Y axis -absorbance at 405 nm in

  Fig.1

Table 1 :

 1 Characterization of the cadmium exposure experiment. Each experimental group is designated and its total number of fish, total number of pooled protein samples, average fish weight, average oocyte diameter and detailed treatment description is provided. IP -intra-

	peritoneal.
	Group No.	Number	Fish	Average	Treatment description
		of	of	weight	oocyte
		fish	protein	[gr]	diameter
			samples		[µm]
						31 aliquots of food pellets containing no
	C	8	3	38.8±5.4	67±26	cadmium, each aliquot of 1% body weight, four times a week, sacrificed 3 days after last
						feeding.
						31 aliquots of food pellets containing 3.23 µg
	LF	10	3	39.7±4.2	74±57	cadmium/gr food, each aliquot of 1% body weight, four times a week, sacrificed 3 days
						after last feeding.
						31 aliquots of food pellets containing 57.25 µg
	HF	10	3	36.2±5.1	61±36	cadmium/gr food, each aliquot of 1% body weight, four times a week, sacrificed 3 days
						after last feeding.
	Ix1	9	2	40.4±6.2	70±34	One IP injection of 50 µl Cd in saline (2 mg/ml), sacrifice after 8 days.
						Four IP injections of 50 µl Cd in saline (2
	Ix4	7	1	40.3±4.6	63±30	mg/ml)at 3 days intervals, sacrificed 4 days
						after fourth injection

Table 2 :

 2 Bio-monitoring of the Israel Mediterranean coast. Results are presented as average ± standard deviation. Fish sample size is presented within parentheses.

	Biomarker	Haifa Bay -Polluted site	10-25 km south to Haifa Bay -Relatively clean sites
	Transcript (2004) -Biomarker / Actin [atomol/atomol]
	Metallothionein	0.43±0.40 (20)	0.24±0.13 (8)
	Cytochrome P4501A	2.07±1.48 (20)	0.53±0.25 (8)
	Transcript (2005) -Biomarker / Actin [atomol/atomol]
	Metallothionein	0.79±0.48 (20)	0.31±0.47 (20)
	Cytochrome P4501A	3.84±3.86 (8)	0.54±0.36 (8)
		Protein (2004) [nmol mg -1 total protein]
	Cytochrome P4501A	27.9 ± 19.9 (20)	5.3 ± 4.8 (8)

Fig.4

Acknowledgement

This study was supported by EU GENIPOL project (EVK-2001-00057). Special appreciation is extended to Ms. Maria Segal for her excellent care of the fish.