Antioxidant efficiency in early life stages of the Antarctic silverfish, Pleuragramma antarcticum: Responsiveness to pro-oxidant conditions of platelet ice and chemical exposure

Aquatic Toxicology 75 (2005) 43–52

Antioxidant efficiency in early life stages of the Antarctic silverfish, Pleuragramma antarcticum: Responsiveness to pro-oxidant conditions of platelet ice and chemical exposure Francesco Regoli a,∗ , Marco Nigro b , Maura Benedetti a , Daniele Fattorini a , Stefania Gorbi a a

Istituto di Biologia e Genetica, Universit`a Politecnica delle Marche, Via Ranieri Monte d’Ago, 60100 Ancona, Italy b Dipartimento di Morfologia Umana e Biologia Applicata, Universit` a di Pisa, Italy Received 30 March 2005; received in revised form 28 June 2005; accepted 7 July 2005

Abstract The Antarctic silverfish Pleuragramma antarcticum is a key organism in the ecology of Southern Ocean. Eggs with fully developed yolk-sac embryos and newly hatched larvae have been recently observed to occur in the platelet ice accumulating below the sea-ice layer. This environment has strong pro-oxidant characteristics at the beginning of austral spring, when the rapid growth of algal ice communities, the massive release of nutrients and the photoactivation of dissolved organic carbon and nitrates, all represent important sources for oxyradical formation. Such processes are concentrated in a short period of a few weeks which overlaps with the final development of P. antarcticum in platelet ice. The aim of this work was to characterize the antioxidant system in embryos of P. antarcticum and the responsiveness toward the natural increase of prooxidant conditions in early spring. Considering the lack of ecotoxicological data on this species and its pivotal importance in the ecosystem of Southern Ocean, the sensitivity of its early life stages was also evaluated after laboratory exposures to environmentally relevant doses of benzo(a)pyrene, as a model chemical potentially released from anthropogenic activities. Obtained results revealed a marked temporal increase of antioxidants in embryos of P. antarcticum as adaptive counteracting responses to oxidative conditions of platelet ice. Particularly prompt responses were observed for glutathione metabolism which, however, did not prevent formation of increasing levels of lipid peroxidation products; from the analysis of total oxyradical scavenging capacity (TOSC), the overall efficiency to neutralize peroxyl radicals remained almost constant while slightly lower TOSC values were obtained toward hydroxyl radicals at the end of sampling period. Laboratory exposures to 0.5–5 ␮g/l BaP caused a significant accumulation of this PAH but no significant effects on the activity of cytochrome P450. Antioxidants of exposed embryos showed less marked variations than embryos in field conditions suggesting that the elevated pro-oxidant

∗

Corresponding author. Tel.: +39 071 2204613; fax: +39 071 2204609. E-mail address: [email protected] (F. Regoli).

0166-445X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2005.07.003

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challenge, to which these organisms are naturally adapted, might be responsible for the moderate responsiveness to pro-oxidant chemicals. © 2005 Elsevier B.V. All rights reserved. Keywords: Pleuragramma antarcticum; Platelet ice; Oxidative stress; Early life stages; Antioxidants; Total oxyradical scavenging capacity; Benzo(a)pyrene; Biomarkers

1. Introduction The notothenid fish Pleuragramma antarcticum has a pivotal importance in Antarctic ecosystems. In the adult stage, this is the only pelagic fish, widely distributed and abundant in shelf waters around the continent, and represents a major contribution to the diet of most Antarctic vertebrates such as whales, seals, penguins, flying birds and benthic fish (La Mesa et al., 2004). The entire life cycle of this species occurs in the water column, larvae and juveniles amounting up to 98% of ichthyoplankton in the Ross Sea (Vacchi et al., 2004). Spawning and embryological development of P. antarcticum still remain to be fully elucidated; eggs have not been described and are thought to be pelagic, whereas spawning events have been suggested to occur at the end of austral winter close to stationary coastal polynias with larvae hatching by November and December in coastal waters (Vacchi et al., 2004). In November 2002, embryonated eggs of P. antarcticum were detected at Terra Nova Bay (Vacchi et al., 2004), floating in huge amounts among the platelet ice, flat ice crystals occurring under the sea-ice during early Antarctic spring. The structure of irregularly diskshaped ice platelets provides a large surface area for the growth of algal and microzooplankton communities (Arrigo et al., 1993), which represent an important food source, but also a favourable environment, protected from predation, for the early life stages of several organisms (Gutt, 2002). The presence of embryos of P. antarcticum at the last stage of development in ice platelets demonstrates the crucial importance of the coastal area of Terra Nova Bay and the significant role of sea-ice on the early stages of life cycle of this species which rapidly migrate down the water column after hatching (Vacchi et al., 2004). The early development in platelet ice might challenge P. antarcticum with a rapidly increase of environmental pro-oxidant pressure. At the beginning of Antarctic spring, the lower layer of coastal

sea-ice is characterized by extremely high concentrations of organic matter, phosphates, nitrates, dissolved organic carbon, proteins, lipids, carbohydrates, bacterial biomass and diatoms (Guglielmo et al., 2000). The return of sunlight activates several biological reactions, mobilization of nutrients, development of autotrophic ice communities and increase of photosynthetically produced oxygen in the platelets (G¨unther et al., 1999). Solar irradiance, through the photolysis of dissolved organic matter (DOM) and nitrates, is a major pathway for release of hydrogen peroxide and hydroxyl radicals also in Antarctic seawaters where approximate levels of 30 nM H2 O2 have been detected in coastal and offshore sites (Karl and Resing, 1993; Qian and Kieber, 1995; Abele et al., 1999; Yocis et al., 2000; Qian et al., 2001; Croot et al., 2005). Similar measurements are actually not available for the platelet ice where the sunlight mediated generation of ROS should be enhanced due to the massive release of DOM in November and the oversaturation of oxygen caused by the rapid algal growth (G¨unther et al., 1999; Guglielmo et al., 2000; Delille et al., 2003). All these reactions are concentrated in a relatively short period of 3–6 weeks when the lower layer of sea-ice loses its biological richness before the melting. Considering the potential pro-oxidant characteristics of sea-ice and associated ice platelets, the main objective of this work was to characterize the antioxidant defences and their temporal variations in embryos of P. antarcticum; while the basal levels of oxygen detoxification systems reflect the natural oxidative challenge to which organisms are exposed (Jamieson et al., 1986; Regoli et al., 2000a), the capability to enhance antioxidant efficiency is responsible for the adaptation to rapid changes of environmental pro-oxidant conditions (Brown et al., 2002; Regoli et al., 2004a; Gorbi et al., 2005). The balance between pro-oxidant forces and antioxidant defenses is a matter of growing interest for polar organisms which, compared to similar temperate

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species, are often characterized by more elevated capability in neutralizing the toxic effects of oxyradicals. Various hypotheses have been suggested to explain such features in different organisms, including elevated levels of dissolved oxygen in cold seawater, higher density of mitochondria in fish, algal symbionts in several invertebrates, membrane lipid composition to maintain fluidity at low temperatures, reduced respiration rate to compensate for low food supply and long periods of starvation, marked changes in food availability and feeding activities, efficient long-term protection needed for RNA and proteins with extremely low turnover rate and higher exposure to oxidative stress (Viarengo et al., 1994; Regoli et al., 1997, 2000b, 2002; Johnston et al., 1998; Abele and Puntarulo, 2004; Camus et al., in press). Studies on susceptibility to oxidative stress in key Antarctic organisms are of value also for monitoring the impact of human activities in these remote areas and the development of biomarkers revealing perturbation of the redox status and oxidative pathways of toxicity have been demonstrated toward various classes of chemical pollutants (Regoli et al., 1998, 2005). Due to the ecological importance of P. antarcticum, a second aim of this work was a preliminary evaluation of oxidative sensitivity of early life stages to polycyclic aromatic hydrocarbons (PAH), model chemicals potentially released as a consequence of both normal human activities and during accidental oil spills. The pro-oxidant effects of BaP have already been described in several fish species where oxyradical formation can be, at least partly, associated with induction of cytochrome P450 and occurrence of redoxcycling metabolites (Regoli et al., 2003); on the other hand, low activities of biotransformation enzymes were described in Antarctic fish from Terra Nova Bay with significant effects on both bioaccumulation and biological consequences of PAHs (Regoli et al., 2005). The overall results of this work were expected to provide the first characterization of the antioxidant defences in early life stages of P. antarcticum and the presence of variations possibly reflecting changes of environmental pro-oxidant conditions in the lower layer of sea-ice and ice platelets at the beginning of Austral spring. This study also represents an additional contribution toward the assessment of sensitivity to chemical exposure in key species of the Antarctic marine ecosystem.

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2. Materials and methods 2.1. Sample collection and exposure to BaP During the XIX Italian Antarctic Expedition (2003–2004), early life stages of P. antarcticum were sampled in platelet ice near the Italian Base “Mario Zucchelli” at Terra Nova Bay. The appearance of embryonated eggs was monitored every other day by visually analysing the platelets collected through 1.3 m diameter holes drilled in the 2 m-thick ice (Vacchi et al., 2004). Eggs with yolk-sac embryos and newly hatched larvae were first detected among the platelets in early November 2003 and found only for the following 2 weeks. In this short period, the sampling method prevented an accurate analysis of distribution and abundance of early life stages in the platelets, but a rough estimate proposed also by Vacchi et al. (2004), indicated several thousands of embryonated eggs per hole. Embryos appeared in an advanced stage of development (stage V), just before hatching, completely developed and wrapping around the yolk-sac for more than 360◦ (Fig. 1); morphological features and the pigmentation pattern resembled the detailed description given by Vacchi et al. (2004). Newly hatched larvae were also observed but, since handling and light irradiance could have influenced the hatching (Vacchi et al., 2004), they were not separated from embryos for biochemical analyses; in this respect we might exclude a change in the early life stages population under the ice or a quantitative shift in antioxidant defences from embryos to larvae of P. antarcticum which, after hatching, rapidly move from the sea-ice layer to the water column (Vacchi et al., 2004). Eggs were collected in three occasions (5, 14 and 20 November), gently separated from the platelets, pooled in different samples (0.2–0.5 g each), frozen in liquid nitrogen and maintained at −80 ◦ C. Embryos and larvae obtained during the first collection were also exposed for 24 h to benzo(a)pyrene (previously dissolved in DMSO) at final assay concentrations of 0.5, 1 and 5 ␮g/l; exposures were carried out in glass beakers at the environmental temperature of −1 ± 0.5 ◦ C. 2.2. Biochemical analyses Activities of the antioxidant enzymes (catalase, glutathione reductase, glutathione peroxidases,

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Fig. 1. Embryonated eggs of P. antarcticum at stage V of development, with fully formed embryos wrapping around the yolk-sac for more than 360◦ .

glutathione S-transferases) were determined after homogenization (1:5 w/v ratio) in 100 mM potassium phosphate buffer pH 7.4, 1 mM EDTA, 1.15% KCl and centrifugation at 10,000 × g for 30 min; the resulting supernatants were further centrifuged at 100,000 × g for 1.10 h and measurements carried out by spectrophotometric assays at the constant temperature of 18 ± 1 ◦ C (Regoli et al., 2003, 2005). Catalase (CAT) was measured by the decrease in absorbance at 240 nm (extinction coefficient, ε = 0.04 mM−1 cm−1 ) due to the consumption of hydrogen peroxide, H2 O2 (12 mM H2 O2 in 100 mM K-phosphate buffer pH 7.0). Glutathione reductase (GR) was determined from NADPH oxidation during the reduction of oxidized glutathione, GSSG (λ = 340 nm, ε = −6.22 mM−1 cm−1 ). The final assay conditions were 100 mM K-phosphate buffer pH 7.0, 1 mM GSSG, and 60 ␮M NADPH. Glutathione peroxidases activities were assayed in a coupled enzyme system where NADPH is consumed by glutathione reductase to convert the formed GSSG to its reduced form (GSH). The decrease of absorbance was monitored at 340 nm (ε = −6.22 mM−1 cm−1 ) in 100 mM K-phosphate buffer pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium azide NaN3 (for hydrogen peroxide assay), 2 mM GSH, 1 U glutathione

reductase, 0.24 mM NADPH, and 0.5 mM hydrogen peroxide or 0.8 mM cumene hydroperoxide as substrates, respectively, for the selenium-dependent and for the sum of Se-dependent and -independent forms. The rate of the blank reaction was subtracted from the total rate. Glutathione S-transferases (GST) were determined at 340 nm using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The assay was carried out in 100 mM K-phosphate buffer pH 6.5, 1.5 mM CDNB, 1 mM GSH (ε = 9.6 mM−1 cm−1 ). Total glutathione was analysed in samples homogenized (1:5 w/v ratio) in 5% sulfosalicylic acid with 4 mM EDTA, maintained for 45 min on ice and centrifuged at 37,000 × g for 15 min. The resulting supernatants were enzymatically assayed (Regoli et al., 2003). The total oxyradical scavenging capacity (TOSC) assay measures the overall capability of cellular antioxidants to absorb different forms of artificially generated oxyradicals, thus inhibiting the oxidation of 0.2 mM ␣-keto-␥-methiolbutyric acid (KMBA) to ethylene gas (Winston et al., 1998; Regoli and Winston, 1999). Peroxyl radicals (ROO• ) were generated by the thermal homolysis of 20 mM 2-2 azo-bis-(2-methylpropionamidine)-dihydrochloride (ABAP) in 100 mM K-phosphate buffer, pH 7.4. Hydroxyl radicals (• OH) were generated from

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the Fenton reaction of iron–EDTA (1.8 ␮M Fe3+ , 3.6 ␮M EDTA) plus ascorbate (180 ␮M) in 100 mM K-phosphate buffer. Under these conditions the different oxyradicals produced quantitatively similar yields of ethylene in control reactions, thus allowing comparisons of the relative efficiency of cellular antioxidants toward a quantitatively similar radical flux (Regoli and Winston, 1999). Ethylene formation in control and sample reactions was analyzed at 10–12 min time intervals by gas chromatographic analyses according to Regoli and Winston (1999). The TOSC values from the equation
are quantified

TOSC = 100 − ( SA/ CA × 100), where SA
and CA are the integrated areas calculated under the kinetic curves for samples (SA) and control (CA) reactions, respectively. For all the samples, a specific TOSC (normalized to content of protein) was calculated by dividing the experimental TOSC values by the relative protein concentration contained in the assay. For the analysis of ethoxyresorufin O-deethylase (EROD), samples were homogenized (1:5 w/v ratio) in 100 mM K-phosphate buffer pH 7.4, 1 mM EDTA, 1.15% KCl and centrifuged at 10,000 × g for 30 min. Supernatants were further centrifuged at 100,000 × g for 1.10 h and the resulting pellets (microsomal fractions) immediately resuspended in 100 mM K-phosphate buffer pH 7.4, 1 mM EDTA, 1.15% KCl, 20% glycerol and stored at −80 ◦ C. EROD activity was determined by incubation of microsomes in a final volume of 1 ml containing 100 mM K-phosphate buffer pH 7.5, 4 ␮M 7-ethoxyresorufin and 0.25 mM ␤-nicotinamide adenine dinucleotide (NADPH) for 1 h at 18 ◦ C. Reactions were stopped by the addition of 2 ml acetone, and centrifuged. Incubation mixtures as above but stopped at time zero, were used as blank values and subtracted from the sample fluorescence. Fluorimetric analyses (535/585 nm) were quantified by reference to resorufin standards (0.02–1 ␮M). Levels of lipid peroxidation products were analysed as content of malondealdehyde (MDA) according to Shaw et al. (2004). Levels of MDA were estimated by derivatization with 1-metyl-2-phenylindole and calibrated against a malondealdehyde standard curve. Samples were homogenized (1:3 w/v ratio) in 20 mM tris HCl pH 7.4 and centrifuged at 3000 × g for 20 min. Tissue MDA levels were derivatized in a 1 ml reaction mixture containing a final concentration of 10.3 mM 1-metyl-2-phenylindole (dissolved in

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acetonitrile/methanol 3:1), HCl 32%, 100 ␮l water or sample or standard (standard range 0–6 ␮M 1,1,3,3tetramethoxypropane, in 20 mM Tris–HCl, pH 7.4). The tubes were vortexed and incubated at 45 ◦ C for 40 min. Samples were cooled on ice, centrifuged at 15,000 × g for 10 min and read spectrophotometrically at 586 nm. Results were expressed as MDA nmol/g wet weight. Protein concentrations were determined with the Lowry method by using bovine serum albumin (BSA) as standard. 2.3. Chemical analyses of benzo(a)pyrene Samples were extracted in methanol (1:10 w:v) with microwave (150 W for 10 min) and centrifuged at 600 × g for 5 min; methanolic solutions were concentrated in speedvac (RC1009, Jouan, Nantes, France) and purified with solid phase extraction (Octadecyl C18, 500 mg × 6 ml, Bakerbond, Mallinckrodt Baker, Phillipsburg, NJ, USA). A final volume of 0.5 ml was recovered with acetonitrile and highperformance liquid chromatography (HPLC) analyses with fluorimetric detection were carried out using a water:acetonitrile gradient (40:60% for 2 min; to 100% acetonitrile in 10 min and maintained for 5 min; to 40:60% in 2 min and maintained for 6 min). Benzo(a)pyrene was identified by the retention time of appropriate pure standard solutions and the QA and QC were tested processing blank and references samples (Mussel Tissues Standard SRM 2977, NIST). 2.4. Statistical analyses Analysis of variance (ANOVA) was used to compare antioxidant parameters in different samplings and the effects of BaP exposures. The homogeneity of variance was analysed by Cochran C, and post hoc tests (Newman-Keuls) were used to discriminate between means of values.

3. Results Temporal variations of antioxidant responses in early life stages of P. antarcticum sampled from platelet ice at Terra Nova Bay are reported in Fig. 2. A significant time-course enhancement was observed for all

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Fig. 2. Temporal variations of antioxidant parameters in early life stages of Pleuragramma antarcticum sampled from platelet ice in November 2003. Results are expressed as mean values ± standard deviations (n = 10). The p values are reported for significant variations, and different letters indicate significant differences between groups of means (post hoc comparison).

the enzymes from the beginning of November; higher activities were measured for catalase at the last sampling period and earlier variations were obtained for glutathione S-transferases and for glutathione peroxidases (only Se-dependent forms are shown in Fig. 2). Glutathione reductase varied with a linear increment at different samplings (Fig. 2) and also the content of total

glutathione almost doubled from 0.11 ± 0.01 ␮mol/g, during the analysed time-frame; although individual antioxidants revealed counteracting responses, increasing levels of lipid peroxidation products were also measured in early life stages of P. antarcticum (Fig. 2). The total oxyradical scavenging capacity exhibited almost constant values of 400 UTOSC/mg protein for peroxyl

Fig. 3. Variations of benzo[a]pyrene content, activity of ethoxyresorufin O-deethylase (EROD) and antioxidant parameters in early life stages of Pleuragramma antarcticum exposed to different doses of BaP. Results are expressed as mean values ± standard deviations (n = 10). The p values are reported for significant variations, and different letters indicate significant differences between groups of means (post hoc comparison). b.d.l.: below detection limit.

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radicals, while it slightly (not significantly) lowered at the end of sampling period for hydroxyl radicals (Fig. 2). Laboratory exposures to 0.5–5 pg/l BaP caused a significant accumulation of this PAH with similar values for different treatments (Fig. 3); no significant effects were obtained for the activity of cytochrome P450, with a slight increase of EROD activity only at the highest dose (Fig. 3). Variations of antioxidant responses in treated organisms were more limited as compared to those obtained in natural conditions (Fig. 3). Catalase increased at highest exposure dose when also a marked inhibition of glutathione peroxidases was observed in treated early life stages; no significant variations were measured for other oxidative biomarkers (glutathione S-transferases, glutathione reductase, levels of total glutathione and efficiency to neutralize peroxyl radicals). On the other hand, levels of MDA revealed enhanced lipid peroxidation with a more elevated effect at the lowest BaP exposure dose when also TOSC values toward HO• were lower (Fig. 3).

4. Discussion The capability to rapidly enhance the antioxidant efficiency is a fundamental and widely distributed strategy in those species which normally experience marked changes of environmental pro-oxidant pressure in their life cycle (Jamieson et al., 1986; Gorbi and Regoli, 2003; Regoli et al., 2004a). The results obtained in the present study clearly revealed a significant and rapid increase of antioxidants in early life stages of P. antarcticum associated to platelet ice, supporting the hypothesis of a marked enhancement of oxidative challenge experienced by these organisms at the beginning of Antarctic spring. Particularly prompt responses were observed for glutathione metabolism with increased levels of this scavenger and increased activities of glutathionedependent enzymes. The responsiveness of these defenses in temperate and Antarctic species has already been shown toward natural and anthropogenic stressors such as UV radiation, seasonality of feeding activities, presence of photosynthesizing symbionts, adaptation to diving and anaerobic metabolism, exposure to chemical oxidants and pollutants (Abele

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et al., 1998; Regoli et al., 1998, 2000b, 2002; Abele and Puntarulo, 2004; Regoli et al., 2005). Similarly, catalase is a key antioxidant enzyme that removes hydrogen peroxide, a fundamental mechanism also to limit formation of highly reactive hydroxyl radicals in marine organisms (Regoli et al., 2000b). The induction of catalase in P. antarcticum was observed at the last sampling period and such slower time-course response confirms that individual defenses can vary with different activation sequences and complex interactions between pro-oxidant forces and single antioxidants (Regoli et al., 2003). Biphasic and transitory responses of catalase have been previously linked to the overcoming of antioxidant efficiency and occurrence of oxidative damages (Regoli et al., 2004b); in this respect it is interesting to note that, despite the enhancement of glutathione metabolism, higher levels of lipid peroxidation products (MDA) preceded the induction of catalase activity. Abele et al. (1998) reported increased antioxidant defenses and lipid peroxidation in the Antarctic limpet exposed to hydrogen peroxide and higher activities of catalase and glutathione peroxidases have been shown to reduce the occurrence of lipid peroxidation in mammals and fish (Barja de Quiroga et al., 1989; Rodriguez-Ariza et al., 1993). The antioxidant efficiency of P. antarcticum was further characterized by the Total Oxyradical Scavenging Capacity (TOSC) which summarizes the whole capability to neutralize different oxyradicals in a quantitative index (Winston et al., 1998; Regoli and Winston, 1999). Although individual antioxidants were sensitive in revealing a varied pro-oxidant challenge in platelet ice, these changes did not significantly contribute to enhance the overall resistance toward peroxyl radicals while even lower TOSC values for hydroxyl radicals might contribute to explain the increase of lipid peroxidation products. The apparent discrepancy between responses of individual antioxidants and TOSC values suggest that other non-enzymatic scavengers might significantly contribute to balance the redox status of P. antarcticum, confirming the importance of an integrated approach with both individual antioxidants and TOSC for analysing oxidative stress responses in field conditions (Gorbi and Regoli, 2003). Variations of antioxidant profile of P. antarcticum cannot rule out the possibility of some metabolic or intrinsic change. Relationships between antiox-

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idants and early development have been rarely considered in fish species with only few examples for embryos and larval stages (Aceto et al., 1994; Peters et al., 1994; Peters and Livingstone, 1996; Mourente et al., 1999). These studies generally revealed limited changes during the very early development stages with catalase, superoxide dismutase and glutathione dependent enzymes (GPx, GST and GR) almost constant or even decreased in unhatched embryos and during the first 3–5 days after hatching; larger variations occurred in the following days of larval development with both increases and decreases of different antioxidants. In this study, we cannot exclude some metabolic changes occurring during early development since we did not directly measure the concentration of hydrogen peroxide in the platelet; however, only embryos in stage V, the last before hatching, and newly hatched larvae were found and the almost contemporary and rapid increase of all analysed antioxidants would suggest an environmental challenge more than a physiological compensation. The marked changes and reactions at the lower layer of sea-ice and the photooxidation of DOM in the platelet zone are the most intriguing explanation for the rapid increase of antioxidant efficiency in embryos of P. antarcticum. The elevated pro-oxidant conditions in the platelet zone nursery of these fishes could support the hypothesis for a systematic selection of individuals with ample antioxidant protection or induction capability. The enhancement of antioxidant protection can represent an additional advantage also for the pelagic phase in highly irradiated surfaces after the ice melting; catalase can be inactivated in transparent animals exposed to UVB radiation (Oberm¨uller et al., in press) and solar irradiance prevents the survival of some pelagic species in polar waters (Rautio and Korhola, 2002) where reduced repair mechanisms further increase susceptibility to photochemical damage (Ross and Vincent, 1998). The present study also provided a preliminary ecotoxicological investigation on P. antarcticum a matter of great importance due to the constant increase of human activities and the limited biological knowledge to predict the impact of environmental pollutants on key Antarctic species. The lack of fundamental ecotoxicological data is even greater for the earlydevelopmental stages which compared to temperate species, might be more susceptible to environmental

disturbance which could occur over longer periods than in temperate environments (Chapman and Riddle, 2003). Considering the elevated oxidative responses observed in P. antarcticum in natural field conditions, it was of interest to assess the sensitivity of these organisms toward a pro-oxidant chemical. Benzo(a)pyrene was chosen as a model for poly cyclic aromatic hydrocarbons potentially released at local level by routine activities of scientific stations or as a consequence of unpredictable oil-spill events. Metabolization of PAHs in fish is mostly catalyzed by the induction of cytochrome P450 (CYP1A) which is responsible of the detoxification or activation of aromatic substrates, the occurrence of carcinogenic metabolites and enhancement of various forms of cellular toxicity, including oxidative stress. A few studies investigated the sensitivity of temperate fish larvae to organic xenobiotics under both laboratory and field conditions, suggesting the same potential mechanisms of biotransformation and oxidative toxicity (Peters et al., 1996). Embryos of P. antarcticum exposed to relatively low doses of BaP demonstrated the possibility for this xenobiotic to be accumulated in non-active feeding stages after a short 24 h exposure. On the other hand, the metabolization efficiency of the compound was quite limited, with no significant induction of cytochrome P450 in most exposure concentrations, and only a slight increase at the highest exposure level. While induction of CYP1A has been shown in temperate fish larvae (Peters et al., 1994, 1996; Peters and Livingstone, 1995), reduced activity of biotransformation enzymes was demonstrated in the Antarctic fish Trematomus bernacchii and hypothesized to be a normal feature for fish species from Terra Nova Bay (Regoli et al., 2005). At the moment, our data on P. antarcticum do not allow to speculate about the mechanisms underlying the poor response of cytochrome P450 but certainly the results indicate that the sensitivity of early life stages of Antarctic fish to aromatic pollutants needs to be further investigated. Antioxidants of exposed embryos showed a less marked and different pattern of variations compared to those exhibited in field conditions suggesting that the elevated natural pro-oxidant challenge to which these organisms are adapted might be responsible for the moderate responsiveness to pro-oxidant chemicals. Only catalase significantly increased in exposed P. antarcticum, confirming previous data on early life stages of other fish species

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(Peters et al., 1994; Peters and Livingstone, 1995); on the other hand, the inhibition of glutathione peroxidases and some higher levels of lipid peroxidation processes could reveal an oxidative destabilization of cellular membranes and a compromised capability of exposed embryos to respond in an adaptive manner. In conclusion, the overall results of this work provided the first characterization of antioxidant efficiency in early life stages of P. antarcticum, a key species in the ecology of Antarctic ecosystems. Antioxidants were confirmed to be crucial as adaptive responses to extreme environmental conditions and to the rapid changes of pro-oxidant pressure associated to platelet ice. The utility of antioxidants includes their use as biomarkers of chemical disturbance although the limited metabolism of BaP in early life stages will require future investigations.

Acknowledgments This study was financially supported by the Italian National Program on Antarctic Research (PNRA). We wish to thank Dr. Doris Abele (AWI, Bremerhaven) for her useful suggestions and critical review of the manuscript.

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