Naphthalene treatment alters liver intermediary metabolism and levels of steroid hormones in plasma of rainbow trout (Oncorhynchus mykiss)

ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 66 (2007) 139–147 www.elsevier.com/locate/ecoenv

Naphthalene treatment alters liver intermediary metabolism and levels of steroid hormones in plasma of rainbow trout (Oncorhynchus mykiss) Adria´n Tintos, Manuel Gesto, Jesu´s M. Mı´ guez, Jose´ L. Soengas Laboratorio de Fisioloxı´a Animal, Departamento de Bioloxı´a Funcional e Ciencias da Sau´de, Facultade de Ciencias do Mar, Universidade de Vigo, E-36310 Vigo, Spain Received 27 July 2005; received in revised form 24 November 2005; accepted 30 November 2005 Available online 8 February 2006

Abstract To assess the effects of naphthalene on liver intermediary metabolism and plasma steroid hormones, immature female rainbow trout (Oncorhynchus mykiss), in a first experiment, were intraperitoneally injected (2 mL g1) with vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1) and returned to their tanks. At 1, 3, and 6 h after injection, eight fish were sampled from each group. A second experiment was similarly designed but used fish intraperitoneally implanted (10 mL g1) with slow-release coconut oil implants alone (control) or containing naphthalene at doses of 10 and 50 mg kg1 body weight that were sampled 1, 3, and 5 days after injection. At each sampling time, plasma hormone levels (cortisol and 17b-estradiol) and metabolic parameters in plasma (glucose and lactate) and liver (glucose, lactate, and glycogen levels and HK, GK, GPase, GDH, FBPase, and PK activities) were assessed. Changes described for both hormonal systems resulted in a decrease in plasma levels of cortisol and 17b-estradiol. Changes observed in intermediary metabolism described effects in several pathways of liver energy metabolism due to naphthalene. These changes can be summarized as increased glycogenolysis, use of exogenous glucose, and glycolysis and decreased gluconeogenesis. The increased energy production in liver suggested by these changes can be related to the increased detoxification activity known to occcur in liver after PAH exposure, and can be also related directly or indirectly to the changes observed in the levels of plasma steroids. r 2006 Elsevier Inc. All rights reserved. Keywords: Naphthalene; Cortisol; 17b-Estradiol; Energy metabolism; Rainbow trout

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) constitute one of several classes of organic molecules released into the environment largely as the result of human activities, resulting in the ubiquity of these toxic chemicals in the marine environment (Meador et al., 1995). In fish, PAHs have been demonstrated to be mutagenic and carcinogenic precursors as well as to impair growth, reproduction, and osmoregulation (Nicolas, 1999). PAHs interfere with Abbreviations: Asp-AT, Aspartate aminotransferase (EC. 2.6.1.1.); HK, Hexokinase (EC. 2.7.1.11.); FBPase, Fructose 1, 6-bisphosphatase (EC. 3.1.3.11.); GDH, Glutamate dehydrogenase (EC. 1.4.1.2.); GK, Glucokinase (EC. 2.7.1.2.); GPase, Glycogen phosphorylase (EC. 2.4.1.1.); LDH, Lactate dehydrogenase (EC. 1.1.1.27.); PK, Pyruvate kinase (EC. 2.7.1.40.) Corresponding author. Fax: +34 986 812 556. E-mail address: [email protected] (J.L. Soengas). 0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.11.008

endocrine function by either mimicking or blocking the effects of naturally occurring hormones (Johnson et al., 1998). At least some of those alterations appear to occur through changes in the hypothalamus–pituitary–interrenal (HPI) and hypothalamus–pituitary–gonad (HPG) axes (Monteiro et al., 2000b; Zhou et al., 2000). Recent studies in fish suggest that PAHs activate the aryl hydrocarbon receptor (AhR), which then interacts with the estrogen receptor (ER)-dependent pathway (Aluru et al., 2005; Navas and Segner, 2000). Accordingly, PAHs could act as an ER agonist or antagonist and could produce an unbalanced estrogenic response in a target tissue and affect estrogen binding. However, there are conflicting reports regarding whether or not levels of plasma 17b-estradiol (E2) decrease or increase after PAH exposure (Monteiro et al., 2000a; Navas et al., 2004). Cortisol, an energy-mobilizing catabolic hormone, is part of a generalized stress response in fish after xenobiotic

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(including PAHs) exposure (Hontela, 2005). In a way similar to that addressed for estrogens, several PAHs could affect cortisol action, producing either alterations in plasma cortisol levels (Hontela et al., 1992; Pacheco and Santos, 2001a; Teles et al., 2003) or in physiological processes regulated by cortisol, such as energy metabolism, reproduction, growth, and immune function (Hontela, 2005). A number of chemicals, including PAHs, are able to interact with fish endocrine systems which can lead to a disturbance of hormone metabolism or hormone-regulated cellular and physiological processes (Navas et al., 2004). One of those physiological processes could be energy metabolism, since hormones that can be affected by PAH exposure, such as E2 and cortisol, are also known to regulate energy metabolism in fish (Laiz–Carrio´n et al., 2002, 2003; Mommsen et al., 1999; SangiaoAlvarellos et al., 2005a, b). Moreover, fish readily take up lipophilic organic contaminants such as PAHs and possess a variety of cellular mechanisms for protection against the toxic effects of such chemicals (Teles et al., 2003). These processes are energy-expensive and, in theory, should also affect intermediary metabolism of the fish. However, there are few studies addressing the impact of PAHs on energy metabolism in fish. Diesel oil watersoluble fractions (DWSF) are known to increase oxygen consumption in fish (Davison et al., 1992, 1993), whereas changes in plasma metabolites after PAH exposure have been assessed in a few studies, such as for glucose and lactate (Teles et al., 2004, 2005) and lipids (Monteiro et al., 2000a). Studies carried out to assess the impact of PAH exposure on liver energy metabolism are scarce (Roche et al., 2002; Stephensen et al., 2003; Vijayan et al., 1997a). Most of the studies regarding effects of PAHs on fish physiology were carried out using PAH-like substances, such as b-naphthoflavone. However, naphthalene, the simplest PAH, and its methyl-substituted derivatives are the most frequently encountered in PAH-polluted waters (Meador et al., 1995). There are several studies on the effects of naphthalene on biotransformation, genotoxicity, and oxidative stress (Ahmad et al., 2002; Evanson and Van der Kraak, 2001; Gravato and Santos, 2002; Pacheco and Santos, 2002). However, there is little information regarding the effects of naphthalene on steroid endocrine systems (Evanson and Van der Kraak, 2001; Teles et al., 2003), and especially energy metabolism, for which only increased plasma glucose in striped mullet (Thomas and Neff, 1985) and tilapia (Dange, 1986) and decreased liver and muscle glycogen levels in tilapia (Dange, 1986) have been reported hitherto. Therefore, the specific objective of the present study was to examine whether or not naphthalene treatment affects hepatic intermediary metabolism and levels of plasma steroid hormones in fish, using as a model the rainbow trout.

2. Materials and methods 2.1. Fish Sexually immature female rainbow trout (Oncorhynchus mykiss, 6075 g body weight) were provided by a hatchery in Soutorredondo (Noia, Spain) and transferred to the laboratory in the Faculty of Marine Science (Vigo, Spain). Fish were acclimated for 4 wk in flow-through tanks providing a constant supply of fresh well water before the experiments. Fish were kept under natural photoperiod (October 2004) and at constant temperature (15 1C). Fish were fed once daily to satiation with commercial dry pellets (Dibaq-Diprotg SA, Segovia, Spain); proximate food analysis was 48% crude protein, 6% carbohydrates, 25% crude fat, and 11.5% ash; 20.2 MJ kg1 of feed) and were fasted 24 h before sampling. In addition, common water quality criteria were assessed, with no major changes being observed throughout the experiments. The experiments described comply with the Guidelines of the European Union Council (86/ 609/EU) for the use of laboratory animals.

2.2. Experimental design In a first experiment, fish of three different groups (reared in separate tanks) were caught by netting, anaesthetized with MS-222 (50 mg L1) buffered with sodium bicarbonate, weighed, and intraperitoneally injected (2 mL g1) with vegetable (sunflower) oil alone (control) or containing naphthalene (10 and 50 mg kg1) and returned to their tanks. At 1, 3, and 6 h after injection, eight fish were sampled from each group. A second experiment was similarly designed but used fish intraperitoneally implanted (10 mL g1) with slow-release coconut oil implants alone (control) or containing naphthalene at doses of 10 and 50 mg kg1 body weight, and were sampled at 1, 3, and 5 days after injection. In each experiment, uninjected fish were also used to assess the vehicle effect. Fish density in experimental tanks was 5 kg m3. The administration procedure used in the present study was similar to others described in the literature. PAHs dissolved in oil or in lipophylic substances have been used in rainbow trout (Vijayan et al., 1997a; Wilson et al., 1998) and eel (Pacheco and Santos, 1998). Moreover, administration through intraperitoneal injection has been widely used in the literature for PAH delivery (Hutz et al., 1999; Pacheco and Santos, 1998; Vijayan et al., 1997a). Furthermore, the doses of naphthalene used were also similar to those used in other studies with i.p. injections of PAHs (Hutz et al., 1999; Vijayan et al., 1997a; Wilson et al., 1998).

2.3. Sampling At each sampling time, fish were caught quickly by netting and anaesthetized with MS-222 (50 mg L1) buffered with sodium bicarbonate. Blood was obtained in ammonium–heparin treated syringes by puncture of the caudal veins. Plasma samples were obtained by centrifugation of blood and divided into two aliquots. One aliquot was immediately frozen on liquid nitrogen for the assessment of plasma hormone levels, whereas the other aliquot, for the assessment of plasma metabolites, was deproteinized immediately with 0.6 N perchloric acid, neutralized with 1 mol L1 potassium bicarbonate, frozen in liquid nitrogen, and stored at 80 1C until further assay. Liver was removed quickly from each fish, freeze-clamped in liquid nitrogen, and stored at 80 1C until assay.

2.4. Analytical techniques Plasma cortisol levels were measured in plasma by indirect enzyme immunoassay (ELISA) validated for rainbow trout (Tintos et al., 2006). The ELISA satisfied the criteria of specificity (testing cross-reactivity with other steroids), reproducibility (interassay coefficient of variation o6%), precision (intrassay coefficient of variation o4%), and accuracy (average recovery 498%).

ARTICLE IN PRESS A. Tintos et al. / Ecotoxicology and Environmental Safety 66 (2007) 139–147 17b-Estradiol levels were measured in plasma by indirect enzyme immunoassay (ELISA). Briefly, Covalink microplates (Nunc) pretreated with disuccinimidyl suberate were coated with 780 pmol mL1 of a conjugate of bovine serum albumin with the active ester of 6carboxymethyl oxime prepared with 17b-estradiol (Sigma, E-5630). After incubation and blocking with BSA, competition was started by addition of samples and anti-17b-estradiol antibody raised in rabbit (Sigma, E-2885). Goat anti-rabbit IgG conjugated-peroxidase was added as a second antibody (Sigma, A-6154) and then incubated with O-phenylenediamide dihydrochloride (OPD) as substrate. The reaction was stopped with 0.1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear (logit/log) from the lower limit of sensitivity of the assay (0.01 ng mL1) to approximately 100 ng mL1. Dose–response inhibition curves using serially diluted plasma samples consistently showed parallelism with the standard curve using 17b-estradiol. The ELISA satisfied the criteria of specificity (testing cross-reactivity with other steroids: 4.8% estrone, 1.3% estriol, and o0.1% for cortisol, progesterone, corticosterone, and cortisone), reproducibility (interassay coefficient of variation o7%), precision (intrassay coefficient of variation o4%), and accuracy (average recovery 495%). Plasma glucose and lactate were measured using commercial kits from Spinreact (Spain). The frozen liver was finely minced on an ice-cooled Petri dish, vigorously mixed, and divided into two aliquots to assess enzyme activities and metabolite levels. The frozen tissue used for the assessment of metabolite levels was homogenized by ultrasonic disruption with 7.5 vols of ice-cooled 0.6 N perchloric acid, neutralized (using 7.5 vols of 1 mol L1 potassium bicarbonate), centrifuged (2 min at 13,000g, Eppendorf 5415R), and the supernatant used to assay tissue metabolites. Liver lactate levels were determined spectrophotometrically using a commercial kit (Spinreact, Spain). Liver glycogen levels were assessed using the method of Keppler and Decker (1974). Glucose obtained after glycogen breakdown (after subtracting free glucose levels) was determined with a commercial kit (Spinreact, Spain). The aliquots of liver used for the assessment of enzyme activities were homogenized by ultrasonic disruption with 10 vols of ice-cold stopping-buffer containing 50 mmol L1 imidazole-HCl (pH 7.5), 1 mmol L1 2-mercaptoethanol, 50 mmol L1 NaF, 4 mmol L1 EDTA, 120 mmol L1 sucrose, and a protease inhibitor cocktail (Sigma, P-2714). The homogenate was centrifuged (2 min at 13,000g, Eppendorf 5415R) and the supernatant used in enzyme assays. Enzyme activities were determined using a Unicam UV-2 spectrophotometer (Thermo Unicam, Waltham, USA). Reaction rates of enzymes were determined by the increase or decrease in absorbance of NAD(P)H at 340 nm. The reactions were started by the addition of homogenates (0.05 mL) at a pre-established protein concentration, omitting the substrate in control cuvettes (final volume 1.35 mL), and allowing the reactions to proceed at 15 1C for pre-established times (5–15 min). No changes were found in tissue protein levels in any of the groups studied. Therefore, enzyme activities are expressed per mg protein. Protein was assayed in triplicate in homogenates according to Bradford (1976) with bovine serum albumin (Sigma, USA) as standard. Enzymatic analyses were carried out at conditions meeting requirements for optimal velocities. The specific conditions for enzymes were described previously (Sangiao-Alvarellos et al., 2003, 2004, 2005a, b).

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uninjected fish and those fish implanted with vehicle alone (control), either for plasma hormone levels or for the metabolic parameters assessed (data not shown). Plama levels of cortisol (Fig. 1) and 17b-estradiol (Fig. 2) decreased after naphthalene exposure after either short- or long-term treatment. Plasma glucose (Fig. 3) and lactate (Fig. 4) levels displayed dose-dependent increases after short- or longterm naphtahene exposure, with the increase being more marked at short-term for glucose and at long-term for lactate. Changes observed in liver metabolic parameters after short-term (1–6 h) naphthalene exposure are shown in Table 1. Dose-dependent decreases caused by naphthalene exposure were noticed in the different times assessed for glycogen and GDH activity, whereas FBPase activity decreased after 6 h of treatment. Naphthalane treatment produced a dose-dependent increase (for all times assessed) in glucose and lactate levels (though in this later case only after 1 and 3 h of treatment). Similar dose-dependent increases were noticed for GPase activity (for both total activity and the percentage of the enzyme in the active form), PK (for both total activity and the activity ratio of the enzyme), HK and GK activities.

2.5. Statistics The effect of different doses of naphthalene and time of exposure, as well as their possible interaction in parameters assessed, was analyzed using a two-way ANOVA. Multiple comparisons were carried out using the Student–Newman–Keuls test. Significance level was set at Po0:05.

3. Results No mortality, health disturbances, or alterations in behavior were observed in any group of fish throughout the experiments. No differences were observed between

Fig. 1. Changes in the levels of cortisol in plasma of rainbow trout (A) after treatment with 2 mL g1 of vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 6 h after injection, and (B) after treatment with 10 ml g1 of coconut oil implants alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 5 days after injection. Each value is the mean7SEM of 8 fish. *Significantly different (Po0:05) from control fish. # Significantly different (Po0:05) from fish treated with 10 mg naphthalene kg1 body weight. Different letters indicate significant differences among times within each treatment.

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Fig. 2. Changes in the levels of 17b-estradiol in plasma of rainbow trout (A) after treatment with 2 mL g1 of vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 6 h after injection, and (B) after treatment with 10 mL g1 of coconut oil implants alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) that were sampled 1, 3, and 5 days after injection. Further details as in legend of Fig. 1.

Changes observed in liver metabolic parameters after long-term (1–5 days) naphthalene exposure are shown in Table 2. Dose-dependent decreases caused by naphthalene exposure were noticed in glycogen levels and FBPase and GDH activities (in the case of FBPase, the decrease was not observed after 1 day of treatment). In contrast, dosedependent increases caused by naphthalene were noticed in GPase activity (either total activity or the percentage of the enzyme in the active form, though in the later case this did not occur after 1 day of treatment) and PK activity (either total activity or the activity ratio of the enzyme), as well as HK and GK activities.

4. Discussion In the current study, naphthalene-exposed rainbow trout displayed decreased levels of plasma cortisol. This is in agreement with other studies carried out with short-term exposures (3 h–3 days) to naphthalene (Teles et al., 2003) and waters polluted with PAHs (Hontela et al., 1992; Pacheco and Santos, 2001b). However, in other studies no changes in plasma cortisol levels were observed after exposure to phenantrene (Monteiro et al., 2000a), and bnaphthoflavone (Teles et al., 2004, 2005; Wilson et al., 1998; ), and even increases were observed after exposure to naphthalene in killifish (Levitan and Taylor, 1979) and

Fig. 3. Changes in the levels of glucose in plasma of rainbow trout (A) after treatment with 2 mL g1 of vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 6 h after injection, and (B) after treatment with 10 mL g1 of coconut oil implants alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 5 days after injection. Further details as in legend of Fig. 1.

striped mullet (Thomas and Neff, 1985). The decrease in plasma cortisol after naphthalene exposure may be caused by a dysfunction of the HPI axis, in agreement with that suggested by other authors (Monteiro et al., 2000a), and supports the hypothesis of an interrenal secretion impairment generated by exposure to PAHs (Hontela, 2005; Hontela et al., 1992; Pacheco and Santos, 2001a; Teles et al., 2005). However, naphthalene effects can be also attributed to actions on steroidogenesis or breakdown of steroids. The levels of 17b-estradiol also showed a dose-dependent decrease in naphthalene-exposed rainbow trout. Several reports have shown decreased plasma E2 levels in female fish after treatment with naphthalene (Thomas, 1990), phenantrene (Monteiro et al., 2000a,b), benzo(a)pyrene (Monteiro et al., 2000b), b-naphthoflavone (Afonso et al., 1997; Hutz et al., 1999), and waters polluted with PAHs (Johnson et al., 1998). In contrast, increases were also noted after treatment with waters polluted with PAHs (Janssen et al., 1997) or b-naphthoflavone (Navas et al., 2004). The mechanisms by which naphthalene reduced E2 secretion remain to be elucidated, although a number of possibilities, such as inhibition of hypothalamic releasing hormones or pituitary hormones, decreased gonadal cholesterol, stimulation of hepatic catabolism and excretion, or decreased activity of steroidogenic enzymes are

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Fig. 4. Changes in the levels of lactate in plasma of rainbow trout (A) after treatment with 2 mL g1 of vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 6 h after injection, and (B) after treatment with 10 mL g1 of coconut oil implants alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled at 1, 3, and 5 days after injection. Further details as in legend of Fig. 1.

plausible (Afonso et al., 1997). We have no data regarding the cause for the present decrease, but other studies in fish using other PAH-like substances (b-naphthoflavone) suggested a disruptive action upon the HPG axis through the disappearance of the negative feedback control on LH release (Anderson et al., 1996; Navas et al., 2004). For both hormone systems the possible involvement of naphthalene as a disrupter of HPI and HPG axes clearly deserves further experiments regarding the status of hypothalamic and pituitary factors in naphthalene-exposed fish. Changes in the levels of both steroid hormones potentially lead to a disturbance of steroid-regulated cellular and physiological processes in fish. Considering that both hormones are known to produce changes in fish intermediary metabolism (Laiz-Carrio´n et al., 2002, 2003; Sangiao-Alvarellos et al., 2005b), variations in energy metabolism could be expected in naphthalene-exposed fish due to changes in levels of both hormones. On the other hand, intoxication of fish with naphthalene is known to result in increased activation of hepatic biotransformation enzymes such as catalase, GST, and EROD (Pacheco and Santos, 2002; Teles et al., 2003). Thus, in theory, the activation of liver energy metabolism could also result in alterations in intermediary metabolism to provide fuel for those processes.

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Changes observed in glucose and lactate levels in plasma support an enhanced availability of fuels in naphthaleneexposed fish. The increase in plasma glucose is in agreement with the general metabolic stress response observed in other studies after exposure to naphthalene (Dange, 1986; Thomas and Neff, 1985) and other PAHs (Pacheco and Santos, 2001b, 2002; Teles et al., 2004, 2005; Vijayan et al., 1997a). As for lactate, there are no studies carried out with naphthalene, though increases were noticed in fish exposed to DWSF (Pacheco and Santos, 2001a) and waters polluted with PAHs (Teles et al., 2004). In the present study, plasma glucose increased after naphthalene exposure, despite the decreased plasma cortisol levels. Vijayan et al. (1997b) stated that besides interrenal cortisol release, other endocrine systems such as catecholamines might be controlling glucose availability in fish. However, this explanation cannot be applied to the current glucose response since the persistence of catecholamines effects up to 48 h is not likely. Accordingly, the current plasma glucose increase observed following naphthalene exposure may be related to an increased liver gluconeogenesis induced by the previously uptaken plasma cortisol as suggested by Vijayan et al. (1997a). The liver serves as primary receiving organ to i.p. administered PAHs (Deb et al., 2000; Pritchard and Bend, 1984). Accordingly, hepatotoxicity evidence following naphthalene exposure has been reported in humans, based on elevated activities of Asp-AT and LDH (Kurz, 1987). Moreover, PAHs are known to induce in fish liver a general metabolic increase, as a stress response, as has been observed in several fish species, by an enhancement of the transamination capacity (Gravato and Santos, 2002; Pacheco and Santos, 2001b) as well as by an enhancement of the antioxidant and detoxification systems (Anderson et al., 1996; Pacheco and Santos, 1998, 2001a, b, 2002; Teles et al., 2003). These toxic effects, together with changes observed in hormone levels, could produce changes in energy metabolism in fish liver after PAHs exposure. An enhanced glycogenolytic potential was observed in livers of naphthalene-exposed fish based on decreased glycogen levels and increased GPase activity (both total activity and the percentage of the enzyme in the active form). The mobilization of liver glycogen results in glycosyl units ready to be used within liver as demonstrate the simultaneous rise of liver glucose levels. There are few studies in literature to compare with these data, and very few of them were carried out with naphthalene. Thus, a decrease was noticed in liver glycogen levels of tilapia exposed to naphthalene (Dange, 1986), whereas no changes in liver glycogen and glucose levels were observed in rainbow trout intraperitoneally injected with b-naphthoflavone (Vijayan et al., 1997a). In none of those studies was the activity of glycogenolytic enzymes assessed. The capacity of liver for use of exogenous glucose appears to increase due to naphthalene treatment, based on increased HK and GK activities. The increase in the activity of both enzymes occurs simultaneously with the increased levels of

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Table 1 Changes in the levels of metabolites and enzyme activities in liver of rainbow trout after treatment with 2 mL g1 of vegetable oil alone (control) or containing naphthalene (10 and 50 mg kg1 body weight), sampled of 1, 3, and 6 h after injection Parameter

Treatment

Time after treatment (h) 1

3

6

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

18378.30a 16077.84*a 10874.10*#a

19079.41a 16178.78*a 11676.63*#a

185710.5a 15276.52*a 10679.06 *#a

Glucose (mmol g1 body weight)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

32.171.50a 38.973.81*a 41.773.62*a

29.672.09a 33.073.11b 40.772.07*#a

30.272.01a % 37.172.78ab 37.872.89*a

Lactate (mmol g1 body weight)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

3.3170.18a 3.8670.21*a 4.1970.35*a

3.4270.15a 4.7170.34*b 4.7970.28*a

3.2970.19a 3.3570.32a% 3.7570.27a%

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.8270.04a 0.9870.06*a 0.9970.08*a

0.7870.06a 0.9970.05*a 1.1170.08*ab

0.8570.08a % 1.2970.07*b 1.3670.09*b

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

30.371.71a 35.472.13*a 38.171.69*a

31.572.52a 36.371.38a 39.471.99*a

29.771.90a % 43.772.38*a 49.572.31*b

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

3.5170.11a 3.8170.21a 3.9370.20*a

3.4770.17a 3.8770.17a 4.0170.18*a

3.4670.16a % 4.2070.14*a 4.3770.16*a

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

12.770.73a 14.370.82a 17.670.37*#a

13.170.45a 19.270.91*b 22.371.25*b

14.070.62a 24.370.71*c 27.471.31*b

HK activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.1270.01a 0.1670.01*ab 0.1670.01*a

0.1170.01a 0.1470.01*a 0.1570.01*a

0.1170.01a % 0.1770.01*b 0.2270.01*#b

GK activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

1.0770.04a 1.2270.12a 4.0770.30*#a

1.1370.10a 1.8470.11*b 6.5070.57*#b

1.0770.04a % 1.6570.17*b 1.5470.08*c

FBPase activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.5670.03a 0.5370.04a 0.5070.03a

0.5470.02a 0.5270.02a 0.4770.04a

0.5570.02a % 0.4270.01*b 0.3370.03*#b

GDH activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

1.0270.03a 0.9170.03*a 0.9070.04*a

0.9870.05a 0.9170.03a 0.8070.04*#b

0.9670.05a % 0.8870.04a 0.7770.03*#b

Metabolites Glycogen (mmol glycosyl units g1 body weight)

Enzyme activities GPase activity Total activity (U mg1 protein)

% GPase a

PK activity Optimal activity (U mg1 protein)

Activity ratio

Note: Each value is the mean7SEM of 8 fish.*Significantly different (Po0:05) from control fish. #Significantly different (Po0:05) from fish treated with 10 mg naphthalene kg1 body weight. Different letters indicate significant differences (Po0:05) among times within each treatment.

glucose in plasma. Altogether, these metabolic changes suggest that naphthalene exposure induces an enhanced use of glucose in the liver either from glycogen stores or from the blood stream. The enhanced availability of glucose in naphthaleneexposed fish appears to be used in situ through glycolysis, since increased PK activity was observed in naphthaleneexposed fish. There are no studies in literature carried out with the effects of naphthalene in this pathway, though in rainbow trout i.p. injected with b-naphthoflavone (Vijayan

et al., 1997a) no changes were noticed in PK activity. Moreover, decreased gluconeogenic capacity (as suggested by decreased FPBase activity) was also observed in naphthalene-exposed fish in the present study. Again, there are no other studies in the literature regarding naphthalene effects in this pathway, though the decreased gluconeogenic capacity is in agreement with decreased PEPCK activity observed in liver of rainbow trout after bnaphthoflavone treatment (Vijayan et al., 1997a). Since cortisol levels are known to stimulate gluconeogenesis in

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Table 2 Changes in the levels of metabolites and enzyme activities in liver of rainbow trout after treatment with 10 mL g1 coconut oil implants alone (control) or containing naphthalene (10 and 50 mg kg1 body weight) sampled of 1, 3, and 5 days after injection Parameter

Treatment

Time after treatment (days) 1

3

5

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

19578.40a 14674.97*a 10773.09*#a

18379.28a 96.974.75*b 70.475.11*#b

179710.3a 78.774.47*b 38.474.49*#c

Glucose (mmol g1 body weight)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

33.372.81a 43.172.47*ab 45.672.90*a

30.171.38a 52.173.79*a 74.673.88*#b

28.472.51a 39.271.76*b 54.474.11*#a

Lactate (mmol g1 body weight)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

3.2870.14a 3.4370.31a 4.4070.37*#a

3.2970.12a 3.9170.32*a 6.2770.54*#b

3.3370.16a 3.3770.28a 4.2570.24*#a

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.8370.06a 0.9270.08a 1.0670.06a

0.7970.05a 0.9270.06a 1.1770.05*#a

0.8170.07a 1.1470.07*b 1.3870.04*#b

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

32.172.32a 36.471.41a 39.272.38*a

28.471.37a 37.271.66*a 42.372.55*a

31.371.47a 42.771.90*b 51.472.11*#b

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

3.4170.18a 3.7870.24a 3.8970.17*a

3.5370.21a 3.8470.14a 4.2170.12*ab

3.5770.14a 4.5670.22*b 4.6270.19*b

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

15.170.73a 17.471.13a 20.370.74*a

13.670.84a 22.570.71*b 22.470.66*a

14.270.91a 25.770.81*c 26.771.13*b

HK activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.1470.005a 0.1770.008*a 0.2870.01*#a

0.1370.006a 0.1670.007*a 0.2070.008*#b

0.1270.008a 0.1270.006b 0.1870.008*b

GK activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.9970.07a 1.7470.09*a 4.5670.25*#a

1.1270.08a 2.1470.17*b 5.5170.35*#b

1.0370.07a 2.1870.19*b 4.7470.46*#ab

FBPase activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.5770.03a 0.5370.02a 0.5170.02a

0.6170.03a 0.5270.02a 0.4970.02*a

0.5970.03a 0.4870.02*a 0.4370.02*b

GDH activity (U mg1 protein)

Control Naphthalene 10 mg kg1 Naphthalene 50 mg kg1

0.9770.03a 0.9070.03a 0.7770.04*#a

1.0370.03a 0.8270.04*a 0.7570.03*a

0.9570.02a 0.7670.04*a 0.7170.06*a

Metabolites Glycogen (mmol glycosyl units g1 body weight)

Enzyme activities GPase activity Total activity (U mg–1 protein)

% GPase a

PK activity Optimal activity (U mg1 protein)

Activity ratio

Note: Each value is the mean7SEM of 8 fish. *Significantly different (Po0:05) from control fish. #Significantly different (Po0:05) from fish treated with 10 mg naphthalene kg1 body weight. Different letters indicate significant differences (Po0:05) among times within each treatment.

fish liver (Mommsen et al., 1999), the decreased gluconeogenic capacity of rainbow trout liver exposed to naphthalene could be partially attributed to the decrease observed in cortisol levels in plasma of naphthalene-exposed fish. We have also observed a decreased capacity for oxidation of amino acids in liver of naphathalene-exposed fish based on decreased GDH activity. Since ketoacids obtained from oxidation of amino acids can be used mainly through gluconeogenesis this is also supporting the decreased gluconeogenic capacity reported above. Furthermore, since

gluconeogenic enzymes are important in the metabolic adjustments associated with stress, the decrease in this pathway may impair the animal’s ability to elicit a metabolic response to stress. The increased energy production from glucose observed in liver in the present study can be also related to enhanced use within liver to support the increased activities of enzymes involved in oxidative stress and detoxification already demonstrated in livers of fish after treatment with naphthalene (Pacheco and Santos, 2002; Teles et al., 2003)

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and other PAHs (Anderson et al., 1996; Pacheco and Santos, 1998, 2001a, b). Another possible use of the increased production of glucose in liver of naphthalenetreated fish could be the pentose phosphate shunt based on the increased G6PDH activity reported by Stephensen et al. (2003) after exposure of fish to PAHs. An increase in the activity of this pathway would provide reducing power to be used in protection during oxidative stress. Since cortisol and E2 levels decreased in plasma of naphthalene-treated fish, changes in liver energy metabolism could be attributed to a previous effect of those hormones, as suggested for plasma metabolites (see above), or to the action of other hormones. The lack of action of E2 or cortisol could produce effects opposite to those observed in studies assessing the effects of those hormones (LaizCarrio´n et al., 2002, 2003; Sangiao-Alvarellos et al., 2005b). This can be true in some cases (decreased gluconeogenic capacity) but not in others (decreased glycogen levels), and thus decreased levels of cortisol and E2 do not match some of the present results. It is interesting to remark that in mammals the intoxication with 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), a dioxin that binds to Ah-R like PAHs, produces decreased liver gluconeogenesis, and glycogen levels, as well as decreased plasma levels of insulin and corticosterone, and increased glucagon (Viluksela et al., 1999). Changes in glycogenolysis, gluconeogenesis, and corticosteroid levels coincide with those observed in the present study. Therefore, involvement of other hormones on metabolic changes after naphthalene exposure cannot be excluded. In summary, the results obtained in the present study support a decrease in plasma levels of cortisol and 17bestradiol after naphthalene exposure. Moreover, changes observed in intermediary metabolism described effects of naphthalene in several pathways of liver energy metabolism. These changes can be summarized in an increased use of both glycogen stores and exogenous glucose through glycolysis. The increased energy production deduced from those changes can be related to the increased antioxidant and detoxification activity known to occcur in liver after PAHs exposure and can be also related directly or indirectly to the changes observed in the levels of plasma steroids. Acknowledgments This study was partly supported by Grants VEM200320062 (Ministerio de Ciencia y Tecnologı´ a and FEDER, Spain) and PGIDT04PXIC31208PN (Xunta de Galicia, Spain), to J.L. Soengas, and Grant AGL2004-08137-c0403/ACU (Ministerio de Educacio´n y Ciencia and FEDER, Spain) to J.M. Mı´ guez. References Afonso, L.O.B., Campbell, P.M., Iwama, G.K., Devlin, R.H., Donaldson, E.M., 1997. The effect of the aromatase inhibitor fadrozole and two

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