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Aquatic Toxicology 86 (2008) 341–351

Acute and prolonged stress responses of brain monoaminergic activityand plasma cortisol levels in rainbow trout are modified by PAHs(naphthalene, �-naphthoflavone and benzo(a)pyrene) treatment

Manuel Gesto, Jose L. Soengas, Jesus M. Mıguez ∗Laboratorio de Fisiologıa Animal, Departamento de Biologıa Funcional y CC. de la Salud, Facultad de Biologıa,

Universidade de Vigo, 36310 Vigo, Spain

Received 4 September 2007; received in revised form 13 November 2007; accepted 17 November 2007

bstract

We have investigated if treatment with two different PAHs such as naphthalene (NAP) and benzo(a)pyrene (BaP), and the PAH-like compound �-aphthoflavone (BNF), may modify the stress responses elicited in rainbow trout by acute or prolonged stress stimuli, and the possible involvementf brain monoamines in those responses. Two experiments (acute and prolonged stress) were performed. In the acute stress experiment, fish were.p. injected with vegetable oil alone (control) or oil containing NAP, BNF or BaP (10 mg kg−1), and 72 h after injection fish were acutely stressedy chasing for 15 min. In the prolonged stress experiment, a similar group-design and injection protocol were followed, but fish were submitted toevere confinement stress by maintaining fish under high stock density (70 kg fish mass m−3) for 72 h. The levels of cortisol, glucose and lactateere assayed in plasma. In addition, the contents of dopamine (DA), noradrenaline (NA) and serotonin (5HT), as well as their oxidized amineetabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indoleacetic acid (5HIAA) were assayed in telencephalon, hypothalamus,

reoptic region, optic tectum and brain stem, as well as the pituitary. Both acute and prolonged stress stimuli increased plasma levels of cortisol,hich further increase with NAP and BNF treatments after acute stress. In contrast, cortisol levels of fish exposed to prolonged stress showed a

lear tendency to decrease after the treatment with BNF and BaP. Stress stimuli also increased plasma glucose levels, which were not affected byAHs in acute stressed fish but decreased in fish exposed to prolonged stress. Increased plasma levels of lactate in fish exposed to stress decreasedfter PAHs treatment in acute stress but not in prolonged stress. With respect to monoaminergic systems, major changes induced by both acutend prolonged stress were increases of the metabolites DOPAC and 5HIAA and DOPAC/DA or 5HIAA/5HT ratios in several brain regions. PAHsnduced alterations in the normal responses of monoaminergic systems to stress, with dopaminergic system being the most affected after acute stress,

nd serotonergic system after prolonged stress. Those alterations, especially after prolonged stress, showed certain parallelism with alterations oflasma cortisol levels. Thus, results suggest that in stressed fish PAH effects on plasma cortisol levels (and its derived metabolic actions) could ben part mediated by alterations on the monoaminergic systems at the CNS of rainbow trout.

2007 Elsevier B.V. All rights reserved.

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eywords: Naphthalene; �-Naphthoflavone; Benzo(a)pyrene; Stress; Brain mo

. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in

quatic ecosystems and occur at their highest concentrationsear urban centers of human populations (LaFlamme and Hites,978; Meador et al., 1995). These compounds reach water

∗ Corresponding author at: Departamento de Biologıa Funcional y CC. de laalud, Edificio de Ciencias Experimentales, Facultad de Biologıa, Universidade Vigo, 36310 Vigo, Spain. Tel.: +34 986 812386; fax: +34 986 812556.

E-mail address: jmmiguez@uvigo.es (J.M. Mıguez).

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166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2007.11.014

ines; Cortisol; Rainbow trout

ainly from atmospheric deposition, oil spills, land runoff andomestic/industrial discharges, and are of international concernecause of their noxious action at multiple levels on biologicalystems (Teles et al., 2003).

PAHs have been demonstrated to be mutagenic and carcino-enic precursors (Nicolas, 1999), as well as to impair varioushysiological functions in fish among other organisms. Alter-tions of endocrine systems have been frequently reported,

ometimes resulting in impairments of reproductive processes oresponses to stress (Anderson et al., 1996; Al-Kindi et al., 2000;luru and Vijayan, 2004, 2006). PAHs, like other organic pol-

utants such as polychlorinated biphenyls (PCBs) and dioxins,

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42 M. Gesto et al. / Aquatic

re activators of the aryl hydrocarbon receptor (AhR) signal-ng in fish, which is believed to be responsible for many toxicffects of those substances (Aluru and Vijayan, 2004; Incardonat al., 2005). Elevation of plasma levels of cortisol is part ofgeneralized stress response frequently associated with the

ubsequent elevation of glucose and lactate levels in plasmaMommsen et al., 1999). In teleosts, synthesis of cortisol takeslace in the interrenal tissue and is under the control of theypothalamus–pituitary axis. Changes in interrenal function andonsequently in circulating levels of cortisol may compromisehe ability of fish to cope with stressful situations, diminish-ng their survival chance. PAHs and other organic contaminantsave been reported to disrupt the normal cortisol productionnd/or action, producing alterations either in plasma cortisol lev-ls (Wilson et al., 1998; Teles et al., 2003; Tintos et al., 2007) orn physiological processes regulated by cortisol such as energy

etabolism, reproduction, growth or immune function (Hontela,005; Tintos et al., 2006b, 2007).

On the other hand, PAHs and other aquatic pollutants mayvoke an integrated stress response in fish in addition to theiroxic effects at the cell and tissue levels (Wendelaar Bonga,997). Thus, the distinction between direct toxic effects andhose resulting from integrated hormonal stress responses maye difficult to determine. The combined effect of PAHs and otherype of stressors on the integrated stress response of fish has noteen exhaustively studied to date.

Elevation of plasma levels of cortisol are mainly underhe control of the adrenocorticotropic hormone (ACTH)eleased from the pituitary. ACTH secretion in fish seemso be under stimulatory control of the corticotropin-releasingactor (CRF) produced in the hypothalamus (Wendelaaronga, 1997). Several brain monoamine neurotransmitters,

ncluding noradrenaline (NA), dopamine (DA) and serotonin5-hydroxytryptamine, 5HT) are believed to be involved in theontrol and integration of behavioral and physiological stressesponses in teleost fish (Øverli et al., 1999, 2001). A stimu-atory action of brain 5HT on the HPI axis has been reportedn several studies in fish (Hoglund et al., 2000; Lepage et al.,002). However, the role of brain 5HT system in the controlf the HPI axis is not clear, and different serotonergic pathwayseem to be involved either in initiating or terminating the adreno-ortical stress response in mammals depending on action site ofhose pathways in the brain (Markus et al., 2000). Furthermore,oglund et al. (2002) described opposed effects of a serotoner-ic agonist on plasma cortisol levels of Arctic charr dependingn stress condition of fish. Moreover, central catecholaminergicystems have been suggested to stimulate HPI axis activity insh, as occurs in mammals (Øverli et al., 1999; Hoglund et al.,000).

We hypothesize that exposure to PAHs could alter the hor-onal and metabolic stress responses in fish, and that changes

n brain monoaminergic systems could be involved in this inter-ction. Therefore, we have assessed in this study whether or

ot the treatment with two different PAHs, naphthalene (NAP)nd benzo(a)pyrene (BaP) and the PAH-like compound �-aphthoflavone (BNF), may modify the stress responses inainbow trout induced by acute or prolonged stress stimuli, eval-

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logy 86 (2008) 341–351

ating in the same fish plasma levels of cortisol (as well as thosef glucose and lactate) and the monoamine content in severalrain regions. The PAHs used were chosen on one hand due toheir different potency based on molecular weight and action

echanism (NAP and BaP) (Barron et al., 2004), whereas BNFas chosen since it has been described as a strong PAH-like

gonist of AhR in fish (Aluru and Vijayan, 2006).

. Materials and methods

.1. Animals

Immature rainbow trout (Oncorhynchus mykiss, about 70 gody mass) were obtained from a local farm and transferredo the laboratories at Faculty of Biology in Vigo, Spain. Fishere acclimated for at least two weeks in well-aerated 100 l

anks, with an open water system. During the experiments, fishere maintained under natural photoperiod (October 2006) and

emperature (15 ◦C). Fish were fed daily with (1% body mass)ommercial dry pellets (Dibaq-Diprotg S.A., Segovia, Spain).he experiments described comply with the guidelines of theuropean Union Council (86/609/EU) and the Spanish Govern-ent (RD 1201/2005) for the use of animals in research.

.2. Experimental design and sampling

In a first experiment, 10 fish of 5 different groups (reared ineparate tanks) were caught by netting, anaesthetized with MS-22 (50 mg l−1) buffered with sodium bicarbonate, weighed andntraperitoneally injected (2 �l g−1) with vegetable (sunflower)il alone (unstressed and stressed-control fish) or containingAP, BNF or BaP (10 mg kg−1) and returned to their tanks.ish were kept at a density of 10 kg fish mass m−3 and wereormally fed. 72 h after injection all fish (except the unstressedroup) were acutely stressed by chasing for 15 min. Fish wereacrificed 1 h later for sampling. This experiment is named ascute stress.

A similar group-design and injection protocol were followedn the second experiment, but when fish returned to their tanks, auantity of water was removed until reaching a stressing stock-ng density of 70 kg fish mass m−3 (in order to expose themo a severe confinement stress). All stressed groups (controlnd PAHs-treated) were maintained for 72 h under those high-tocking conditions before being sacrificed for sampling. Thisxperiment is named as prolonged stress.

Sampling followed the same protocol in both experiments.t each sampling time, fish were quickly caught by netting,

nesthetized with MS-222 (50 mg l−1) and sacrificed by decap-tation. Just before sacrifice, blood samples were obtained fromaudal vein in heparinized syringes. Blood was immediately cen-rifuged (15 min, 4000 × g) and plasma stored without delay at

80 ◦C. Brains were immediately dissected out and the follow-ng regions were sampled: telencephalon (excluding olfactory

ulbs), hypothalamus, pituitary gland, preoptic region (includ-ng the optic tract), optic tectum (only left half) and braintem (mainly including the medulla oblongata). All tissues weretored without delay at −80 ◦C until assay.

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.3. Analysis of monoamines and metabolites

The contents of NA, DA, 3,4-dihydroxyphenylacetic acidDOPAC, a major DA metabolite), 5-HT and 5-hydroxyindole--acetic-acid (5-HIAA, a major 5-HT metabolite) werenalyzed by high performance liquid chromatography withlectrochemical detection (HPLC-EC) as previously describedGesto et al., 2006). Tissues were homogenized by ultra-onic disruption in 0.1 ml (pituitary), 0.5 ml (hypothalamus,elencephalon, preoptic region) and 1 ml (optic tectum andrain stem) of mobile phase used in the chromatogra-hy. Homogenates were centrifuged (16,000 × g, 10 min)nd supernatants were diluted with mobile phase prioro HPLC analysis. Dilutions were 1:1 (supernatant:mobilehase) for pituitary, optic tectum, medulla oblongata andreoptic region and 1:2 for hypothalamus and telen-ephalon.

.4. Analysis of plasma cortisol levels

Plasma cortisol levels were measured by ELISA as previ-usly described (Tintos et al., 2006a). Plasma glucose and lactateevels were measured using commercial kits from SpinreactSpain).

.5. Data analyses

Effects of stress on untreated fish were assessed using a t-testomparing control-stressed with basal-unstressed fish. Effectsf PAHs in the response of measured parameters to stress were

ssessed using one-way ANOVA. Post hoc multiple comparisonsmong stressed groups (control, NAP, BNF and BaP) were car-ied out using the Dunnett’s test. Significance level was set at< 0.05. SigmaStat v3.11 statistical software (Systat Software

nc.) was used for the analyses.

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ig. 1. Effects of PAH treatment on the response of plasma cortisol levels to acute ornstressed-basal and control-stressed) or containing naphthalene (NAP), �-naphthoflall fish (except basal group) were exposed to acute (chasing for 15 min, 72 h after injetress stimuli. Each value is the mean ± S.E.M. of 10 fish. Significantly different (#Pontrol group.

logy 86 (2008) 341–351 343

. Results

No mortality, health disturbances or any alterations in behav-or were observed throughout the experiments due to PAHreatment. Several unstressed fish (n = 10) were used as nega-ive control for estimation of basal levels of parameters assessedn plasma and brain.

.1. Plasma parameters

Both acute and prolonged stress induced increases intressed-control (with respect to basal unstressed) cortisol levelsFig. 1). Normal increased post-stress levels of plasma cortisolncreased further with NAP and BNF treatment in fish exposedo acute stress by chasing (Fig. 1). Levels tended also to increasefter BaP treatment, but this change was not significant. In con-rast, cortisol levels were not further elevated in PAH-treatedsh exposed to prolonged stress. In fact, cortisol levels showedclear tendency to diminish after BNF and BaP treatment in

elation to stressed-control fish; the result was significant foraP (Fig. 1).

Plasma levels of glucose were higher in acutely stressed-ontrol fish than in unstressed fish (Fig. 2). High levels of post-tress plasma glucose in control fish were not affected by anyf the PAHs tested in acutely stressed fish (Fig. 2). Control fishxposed to prolonged stress also showed higher levels of plasmalucose than unstressed fish. However, in this experiment, thehree PAHs induced a decrease in glucose levels with respecto stressed control fish, resulting in similar glucose levels asnstressed fish (Fig. 2).

In the acute stress experiment, stressed-control group showedigher plasma lactate levels than unstressed fish. Treatment

ith BNF and BaP decreased plasma lactate levels as com-ared to stressed-control fish (Fig. 3). After prolonged stress,here were no effects of stress or PAHs exposure on lactateevels.

prolonged stress stimuli. Fish were injected with vegetable oil alone (2 �l g−1;vone (BNF) or benzo(a)pyrene (BaP) at a dose of 10 mg kg−1 body mass. Then,ction) or prolonged (kept for 72 h under a high stocking density of 70 kg m−3)< 0.05) from basal unstressed group. Significantly different (*P < 0.05) from

344 M. Gesto et al. / Aquatic Toxicology 86 (2008) 341–351

Fig. 2. Effect of PAH treatment on the response of plasma glucose levels to acute or prolonged stress stimuli. Fish were injected with vegetable oil alone (2 �l g−1;u thofla −1

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nstressed-basal and control-stressed) or containing naphthalene (NAP), �-naphll fish (except basal group) were exposed to acute (chasing for 15 min, 72 h afttress stimuli. Each value is the mean ± S.E.M. of 10 fish. Significantly differeontrol group.

.2. Brain monoamines

Acute and prolonged stress induced changes in monoaminesontent in several brain regions, with the most affected regionseing telencephalon, preoptic region, brain stem and, to a lesserxtent, hypothalamus (Tables 1–3). Major changes observedere increases in the levels of amine metabolites. Thus, DOPAC

evels were increased in telencephalon, preoptic region andrain stem of acutely stressed fish (Table 1), and in braintem of prolonged stress-exposed fish (Table 2). 5HIAA lev-ls were increased in all regions examined in acutely stressedsh, and in telencephalon, hypothalamus, brain stem and optic

ectum of prolonged stress-exposed fish. In many cases, changes

n metabolite content induced the subsequent elevation ofOPAC/DA or 5HIAA/5HT ratios (Figs. 4 and 5). NA levelsecreased in the preoptic region after both acute and prolongedtress (Table 3).

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ig. 3. Effect of PAH treatment on the response of plasma lactate levels to acute or pnstressed-basal and control-stressed) or containing naphthalene (NAP), �-naphthoflall fish (except basal group) were exposed to acute (chasing for 15 min, 72 h after injetress stimuli. Each value is the mean ± S.E.M. of 10 fish. Significantly different (#Pontrol-stressed group.

vone (BNF) or benzo(a)pyrene (BaP) at a dose of 10 mg kg body mass. Then,ction) or prolonged (kept for 72 h under a high stocking density of 70 kg m−3)< 0.05) from basal unstressed group. Significantly different (*P < 0.05) from

Changes induced by acute and prolonged stress stimuli intressed-control fish were altered after PAH treatment in severalrain regions. In acutely stressed fish, PAHs mainly affectedopaminergic system (Table 1 and Fig. 4). Thus, DOPAC con-ent decreased in telencephalon with BNF and in preoptic regionith the three PAHs, whereas it increased in tectum and pituitary

fter BaP treatment. DA levels decreased in hypothalamus andectum with BNF and increased with NAP in the preoptic region.hanges in amine and/or metabolite levels elicited in many cases

ignificant variations in the DOPAC/DA ratio, which decreasedith BNF in telencephalon and with NAP in the preoptic region,hereas increased with BNF in hypothalamus and with BaP in

ectum (Fig. 4). Minor changes were also observed in seroton-

rgic system (Table 1), including decreased levels of 5HIAA inelencephalon, and 5HT in telencephalon and optic tectum afterNF treatment. NAP induced an increase in the 5HT content of

he preoptic region.

rolonged stress stimuli. Fish were injected with vegetable oil alone (2 �l g−1;vone (BNF) or benzo(a)pyrene (BaP) at a dose of 10 mg kg−1 body mass. Then,ction) or prolonged (kept for 72 h under a high stocking density of 70 kg m−3)< 0.05) from basal unstressed group. Significantly different (*P < 0.05) from

M. Gesto et al. / Aquatic Toxicology 86 (2008) 341–351 345

Fig. 4. Effect of PAH treatment on changes induced by acute stress in the DOPAC/DA and 5HIAA/5HT ratios. Fish were injected with vegetable oil alone (2 �l g−1;unstressed-basal and control-stressed) or containing naphthalene (NAP), �-naphthoflavone (BNF) or benzo(a)pyrene (BaP) at a dose of 10 mg kg−1 body mass.Then, all fish (except basal group) were exposed to acute stress by chasing for 15 min (72 h after injection). Each value is the mean ± S.E.M. of 10 fish. Significantlydifferent (#P < 0.05) from basal unstressed group. Significantly different (*P < 0.05) from control-stressed group.

346 M. Gesto et al. / Aquatic Toxicology 86 (2008) 341–351

Fig. 5. Effect of PAH treatment on the changes induced by prolonged stress in the DOPAC/DA and 5HIAA/5HT ratios. Fish were injected with vegetable oil alone(2 �l g−1; unstressed-basal and control-stressed) or containing naphthalene (NAP), �-naphthoflavone (BNF) or benzo(a)pyrene (BaP) at a dose of 10 mg kg−1 bodymass. Then, all fish (except basal group) were exposed to prolonged stress (kept for 72 h under a high stocking density of 70 kg m−3). Each value is the mean ± S.E.M.of 10 fish. Significantly different (#P < 0.05) from basal unstressed group. Significantly different (*P < 0.05) from control-stressed group.

M. Gesto et al. / Aquatic Toxicology 86 (2008) 341–351 347

Table 1Tissue concentrations (ng g−1 wet weight) of DA and 5HT and their metabolites DOPAC and 5HIAA (mean ± S.E.M.) in different brain regions of PAH-injectedrainbow trout exposed to acute stress (chasing for 15 min) 72 h after injection and sacrificed 1 h after stress stimulus

Basal Control NAP BNF BaP

TelencephalonDOPAC 3.77 ± 1.88 6.20 ± 1.16↑ 7.00 ± 1.18 2.42 ± 0.84� 3.68 ± 0.68DA 137.78 ± 19.78 156.98 ± 20.14 149.82 ± 12.19 114.55 ± 10.19 150.58 ± 25.525HIAA 186.28 ± 8.81 336.30 ± 22.63↑ 322.80 ± 22.97 252.63 ± 15.78� 304.70 ± 30.625HT 492.67 ± 25.01 577.69 ± 28.16↑ 559.29 ± 34.92 442.89 ± 38.81� 540.75 ± 54.07

HypothalamusDOPAC 2.34 ± 0.57 3.30 ± 0.59 4.28 ± 0.84 5.54 ± 1.31 1.51 ± 0.38DA 1326.33 ± 96.27 1514.31 ± 121.87 1610.09 ± 127.88 1166.09 ± 100.75� 1474.82 ± 128.635HIAA 122.52 ± 6.59 176.11 ± 8.24↑ 159.32 ± 15.36 155.28 ± 11.20 185.91 ± 15.305HT 1330.24 ± 81.16 1445.07 ± 102.94 1455.41 ± 107.83 1214.65 ± 96.56 1512.21 ± 118.07

Preoptic regionDOPAC 8.11 ± 0.70 11.76 ± 1.15↑ 7.66 ± 0.86� 8.83 ± 0.56� 8.72 ± 0.81�DA 262.71 ± 27.33 209.74 ± 18.87 319.21 ± 39.42� 156.60 ± 17.47 143.96 ± 24.115HIAA 101.68 ± 7.84 121.55 ± 7.39↑ 133.79 ± 6.56 101.72 ± 3.93 104.86 ± 8.575HT 437.45 ± 31.17 367.66 ± 21.64 486.62 ± 40.32� 321.12 ± 25.81 288.74 ± 32.24

Brain stemDOPAC 2.14 ± 0.27 6.22 ± 1.17↑ 4.60 ± 0.99 5.44 ± 0.84 7.20 ± 0.82DA 325.82 ± 12.12 322.50 ± 12.56 298.17 ± 7.39 291.57 ± 15.86 311.17 ± 14.265HIAA 95.73 ± 5.75 121.02 ± 6.05↑ 116.59 ± 6.70 109.66 ± 4.72 114.78 ± 5.825HT 170.42 ± 6.95 165.90 ± 6.84 163.47 ± 8.68 166.09 ± 6.02 162.30 ± 7.52

Optic tectumDOPAC 1.29 ± 0.38 0.96 ± 0.20 1.21 ± 0.28 0.89 ± 0.32 1.90 ± 0.26�DA 72.02 ± 4.54 68.07 ± 2.48 61.08 ± 3.96 51.76 ± 3.49� 57.47 ± 5.195HIAA 62.11 ± 4.17 96.14 ± 5.52↑ 82.89 ± 5.35 78.71 ± 4.99 81.28 ± 5.015HT 216.48 ± 11.47 259.11 ± 14.00↑ 219.27 ± 9.47 205.81 ± 10.95� 220.69 ± 17.99

PituitaryDOPAC 11.93 ± 2.38 17.76 ± 1.93 23.64 ± 3.84 16.31 ± 1.87 32.03 ± 6.29�DA 233.18 ± 31.27 223.79 ± 26.44 270.35 ± 41.08 231.76 ± 29.87 274.38 ± 42.915HIAA 20.97 ± 3.88 32.22 ± 7.12↑ 30.56 ± 5.83 29.05 ± 2.95 43.74 ± 7.045HT 30.21 ± 5.79 20.86 ± 3.87 21.82 ± 2.16 30.64 ± 7.26 38.12 ± 8.07

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After prolonged stress, alterations were more important in theerotonergic system than in the other neuronal systems examinedTable 2). Both NAP and BaP induced decreases in 5HIAA levelsn hypothalamus and brain stem, which generally were accom-anied by decreases of the 5HIAA/5HT ratio (Fig. 5). This ratiolso decreased in telencephalon with the three PAHs withouthanges in the levels of the amine or its metabolite. With respecto the dopaminergic system, only NAP induced a decrease inOPAC levels in the preoptic region and in brain stem (Table 2),

he latter accompanied by a decrease in the DOPAC/DA ratioFig. 5). Finally, a decrease in NA levels in brain stem after BaPreatment was the only PAH-induced change in noradrenergicystem observed in this study (Table 3).

. Discussion

Acute or chronic exposures of fish to organic pollutants elicit

oxic stress responses on their own and alter through different

echanisms plasma cortisol levels (Hontela et al., 1992; Alurund Vijayan, 2006). Increases in plasma cortisol levels afterreatment with PAHs and other organic pollutants have been

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ntrol groups) or containing naphthalene (NAP), �-naphthoflavone (BNF) oras basal. (↑, ↓) Significantly (P < 0.05) higher or lower from basal group. (�,

bserved in various teleost species like striped mullet (Thomast al., 1980), coho salmon (Thomas and Rice, 1987), rainbowrout (Aldegunde et al., 1999; Aluru and Vijayan, 2004; Tintost al., 2008) and Pacific herring (Kennedy and Farrell, 2005),mong others. Decreases in plasma cortisol levels have also beeneported (Hontela et al., 1992; Pacheco and Santos, 2001b; Telest al., 2003).

The mechanism of action of PAHs to elicit toxic stressesponses seems to involve binding to AhR, which regulateshe transcription of several AhR-responsive genes (Hahn, 1998),ncluding some members of the cytochrome P450 family, espe-ially CYP1A, which has been used as a biomarker of PAHsxposure (Buhler and Wang-Buhler, 1998). For instance, recenttudies of Aluru and Vijayan (2004) showed that BNF, a syn-hetic analog of naturally occurring flavonoid compounds and arototypic agonist of AhR, increases plasma cortisol concentra-ion in rainbow trout. Accordingly, we have previously reported

hat BNF but also BaP (an ubiquitous PAH) at the same doseshan those used in the present study induced similar elevationsn plasma cortisol levels 24 and 72 h after intraperitoneal injec-ion (Tintos et al., 2008). Also NAP, the simplest PAH, has been

348 M. Gesto et al. / Aquatic Toxicology 86 (2008) 341–351

Table 2Tissue concentrations (ng g−1 wet weight) of DA and 5HT and their metabolites DOPAC and 5HIAA (mean ± S.E.M.) in different brain regions of PAH-injectedrainbow trout exposed for 72 h to prolonged stress (stocking density of 70 kg m−3) after injection

Basal Control NAP BNF BaP

TelencephalonDOPAC 3.77 ± 1.88 4.27 ± 1.37 2.36 ± 1.46 3.15 ± 0.84 4.17 ± 1.62DA 137.78 ± 19.78 106.55 ± 17.92 97.66 ± 4.84 92.21 ± 11.01 127.40 ± 23.235HIAA 186.28 ± 8.81 307.38 ± 17.17↑ 270.12 ± 18.60 290.39 ± 16.12 280.77 ± 15.485HT 492.67 ± 25.01 501.81 ± 21.94 509.08 ± 22.07 542.02 ± 32.07 562.19 ± 32.47

HypothalamusDOPAC 2.34 ± 0.57 2.97 ± 0.69 2.74 ± 0.70 3.20 ± 0.44 2.23 ± 0.37DA 1326.33 ± 96.27 1331.89 ± 63.87 1312.40 ± 134.37 1406.54 ± 79.71 1385.91 ± 125.845HIAA 122.52 ± 6.59 192.66 ± 12.06↑ 151.27 ± 8.26� 196.43 ± 9.53 167.25 ± 5.93�5HT 1330.24 ± 81.16 1345.01 ± 64.73 1337.14 ± 116.14 1369.94 ± 59.77 1394.80 ± 101.27

Preoptic regionDOPAC 8.11 ± 0.70 9.42 ± 0.65 5.96 ± 0.94� 9.72 ± 0.57 7.95 ± 0.64DA 262.71 ± 27.33 143.93 ± 12.01↓ 161.55 ± 20.39 202.80 ± 37.43 181.42 ± 21.155HIAA 101.68 ± 7.84 111.38 ± 6.37 102.13 ± 9.15 115.53 ± 7.16 100.71 ± 5.725HT 437.45 ± 31.17 303.53 ± 19.73↓ 315.83 ± 27.49 355.71 ± 42.49 305.65 ± 25.93

Brain stemDOPAC 2.14 ± 0.27 4.44 ± 0.83↑ 1.97 ± 0.46� 4.08 ± 0.62 3.63 ± 0.28DA 325.82 ± 12.12 280.76 ± 8.54↓ 289.85 ± 16.46 256.85 ± 13.51 256.40 ± 23.865HIAA 95.73 ± 5.75 121.61 ± 7.55↑ 98.74 ± 4.78� 110.26 ± 6.55 91.06 ± 6.77�5HT 170.42 ± 6.95 154.99 ± 7.85 170.10 ± 7.63 160.01 ± 5.34 148.18 ± 5.91

Optic tectumDOPAC 1.29 ± 0.38 3.81 ± 1.06 3.88 ± 1.23 4.69 ± 0.77 4.98 ± 0.99DA 72.02 ± 4.54 64.33 ± 3.25 68.08 ± 2.88 54.47 ± 5.03 62.96 ± 4.235HIAA 62.11 ± 4.17 82.33 ± 5.86↑ 70.89 ± 3.75 74.48 ± 4.16 78.46 ± 7.015HT 216.48 ± 11.47 225.83 ± 9.12 220.63 ± 7.72 209.46 ± 12.29 213.57 ± 7.57

PituitaryDOPAC 11.93 ± 2.38 13.92 ± 1.33 15.35 ± 1.64 17.12 ± 4.08 15.78 ± 2.45DA 233.18 ± 31.27 248.59 ± 25.18 249.29 ± 27.47 201.60 ± 28.70 245.27 ± 29.715HIAA 20.97 ± 3.88 21.25 ± 2.11 23.37 ± 3.35 30.72 ± 5.71 25.66 ± 5.825HT 30.21 ± 5.79 24.31 ± 3.26 29.95 ± 3.36 30.05 ± 6.63 22.86 ± 4.27

Fish were injected (2 �l g−1) with vegetable oil alone (unstressed and stressed-control groups) or containing naphthalene (NAP), �-naphthoflavone (BNF) orbenzo(a)pyrene (BaP) at a dose of 10 mg kg−1 body mass. Unstressed group is named as basal. (↑, ↓) Significantly (P < 0.05) higher or lower from basal group. (�,�) Significantly (P < 0.05) higher or lower from control group.

Table 3Tissue concentrations (ng g−1 wet weight) of NA (mean ± S.E.M.) in different brain regions of PAH-injected rainbow trout exposed to acute stress (chasing for15 min) 72 h after injection and sacrificed 1 h after stress stimulus, or exposed for 72 h to prolonged stress (stocking density of 70 kg m−3) after injection

Basal Control NAP BNF BaP

Acute stressTelencephalon 642.32 ± 30.59 646.51 ± 33.44 676.30 ± 49.27 549.99 ± 47.63 661.40 ± 57.29Hypothalamus 626.22 ± 71.52 717.31 ± 52.13 689.84 ± 70.23 643.37 ± 38.12 787.79 ± 51.41Preoptic region 378.51 ± 24.58 274.88 ± 24.01↓ 301.07 ± 33.79 249.99 ± 29.79 236.00 ± 20.90Brain stem 244.17 ± 11.06 212.06 ± 11.09 218.66 ± 10.13 208.74 ± 10.94 207.74 ± 13.74Optic tectum 216.74 ± 13.55 205.57 ± 12.21 205.33 ± 16.98 184.99 ± 8.86 198.84 ± 11.92Pituitary 104.63 ± 15.46 104.94 ± 6.59 114.62 ± 10.76 110.79 ± 12.88 124.12 ± 16.79

Prolonged stressTelencephalon 642.32 ± 30.59 634.23 ± 39.44 698.88 ± 35.20 604.48 ± 38.86 615.47 ± 55.01Hypothalamus 626.22 ± 71.52 710.95 ± 48.73 744.46 ± 52.55 681.15 ± 26.69 748.47 ± 44.34Preoptic region 378.51 ± 24.58 279.39 ± 23.69↓ 280.03 ± 22.21 250.98 ± 21.47 288.41 ± 17.42Brain stem 244.17 ± 11.06 226.51 ± 10.25 227.22 ± 14.84 197.10 ± 12.03 176.33 ± 16.78�Optic tectum 216.74 ± 13.55 215.62 ± 7.61 220.35 ± 16.06 195.49 ± 12.52 234.20 ± 22.25Pituitary 104.63 ± 15.46 96.49 ± 13.57 103.41 ± 10.21 100.32 ± 17.02 104.51 ± 12.38

Fish were injected (2 �l g−1) with vegetable oil alone (unstressed and stressed-control groups) or containing naphthalene (NAP), �-naphthoflavone (BNF) orbenzo(a)pyrene (BaP) at a dose of 10 mg kg−1 body mass. Unstressed group is named as basal. (↑, ↓) Significantly (P < 0.05) higher or lower from basal group. (�,�) Significantly (P < 0.05) higher or lower from control group.

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hown to alter levels of plasma steroid hormones in rainbow troutTintos et al., 2007), although an AhR-dependent mechanism hasot yet been defined for this PAH. It has been suggested that AhRctivation effects may be caused by a dysfunction of the brainituitary–interrenal axis leading to changes in steroid biosynthe-is and steroid feedback regulation (Hontela, 2005). However,hile several studies have examined the impact of PAHs on

nterrenal steroid formation and regulation (Aluru and Vijayan,004, 2006), the target sites and mechanisms at brain–pituitaryevel have received little attention.

There are only few studies available regarding the interactiveffects of PAHs and other type of stressors on plasma cortisolevels, and those existing report either stimulatory (Vijayan etl., 1997a) or inhibitory effects of PAHs on cortisol responseHontela et al., 1997; Wilson et al., 1998). For instance, Wilsont al. (1998) found decreased levels of plasma cortisol in BNF-reated fish 0.5–24 h after handling stress for 3 min, whereasijayan et al. (1997a) reported increased levels of plasma cor-

isol in BNF-treated trout 1 h after acute handling stress. Wehowed herein that 1 h after acute stress, plasma cortisol levelsere generally higher in PAH-treated than in control-stressedsh. Since stress itself was able to increase plasma cortisol lev-ls, these data suggest that enhanced plasma cortisol levels inAH-treated fish result from the combination of the stimulatoryffects of the acute stress per se and the exposure to PAHs.

Exposure to prolonged stress (72 h of high stocking den-ity) resulted in higher plasma cortisol levels in stressed-controlhan in unstressed fish. Furthermore, in contrast to the effectsbserved after acute stress, treatment with BNF and BaP tendedo decrease levels of cortisol in control fish, though this decreasettained statistical signification only for BaP. Thus, stressed-ontrol fish responded similarly to both types of stress stimuliith respect to unstressed fish, but in the PAH-treated fish the

esponse varied dramatically. The explanation for the differencesn the response of cortisol levels to acute or prolonged stress inAH-treated fish is not evident. Some authors pointed that pro-onged stress may lead to hyperactivity of pituitary cells, and inurn to the exhaustion of the hypothalamus–pituitary–interrenalxis and production of cortisol (Hontela et al., 1992, 1997;obinet and Feunteun, 2002). More recently, Wilson et al.

1998) suggested the possibility that prolonged exposure to pol-utants may lead to downregulation and/or desensitization of thedrenocorticotropic hormone (ACTH) receptors as a mechanismf suppressed cortisol response in fish. In our case, fish exposedo prolonged stress were forced to cope simultaneously for 72 hith the stress resulting from high stocking density and that due

o PAH exposure. Under those conditions, it is reasonable that aigh HPI axis activation could lead to a desensitization of ACTHeceptors, which was not reached in the case of acutely stressedsh. Vijayan et al. (1997b) also suggested that the polychlo-inated biphenyl 3,3′,4,4′-tetrachlorobiphenyl (TCBP, anotherhR ligand), could enhance cortisol clearance by hepatocytes

n rainbow trout. It cannot be discarded that such a clearance-

nhancing effect occurs with PAHs in our case. Following ourypothesis, another plausible explanation (as occurring in dif-erences observed in brain monoamines) is that PAHs exert aifferential action in the central mechanisms controlling stress

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logy 86 (2008) 341–351 349

esponse, depending on the type and/or duration of the stressor.hat issue will be discussed below.

Increases in plasma glucose levels are also a part of atress response in fish, as a result of the stimulatory effect ofatecholamines and corticosteroids released into plasma dur-ng stress (Wendelaar Bonga, 1997). Moreover, simultaneousncreases in plasma cortisol and glucose levels have been alreadyoticed in rainbow trout exposed to BNF or BaP (Tintos et al.,008), which further support the role of these toxicants in thenduction of a stress response in fish. Accordingly, we observederein increased levels of glucose in plasma after submittingrout to both acute and prolonged stress. In addition, PAH treat-

ent did not affect plasma glucose content after acute stress, buthey decreased in treated fish with respect to stressed-controlsfter prolonged stress, showing some parallelism with corti-ol data. Therefore, it seems that plasma glucose response isirectly related to changes in plasma cortisol levels, in agree-ent with that already reported in fish submitted to different

ind of stressors (Mommsen et al., 1999).Plasma lactate levels were decreased by treatment with both

NF and BaP after acute stress (although being always higherhan those of basal-unstressed fish), and unchanged after pro-onged stress, with levels being similar to those of unstressedsh. Lactate increase has been suggested as a part (in termsf carbohydrate metabolism) of a common pattern of stressesponse in fish besides concomitant increases of plasma glu-ose and cortisol (Hontela et al., 1996; Santos and Pacheco,996; Pacheco and Santos, 2001b). Increases in basal levels ofactate have been already noticed in fish exposed to PAHs (Tintost al., 2007; Tintos et al., 2008) as well as in fish exposed toater-soluble fraction of diesel oil (Pacheco and Santos, 2001b).owever, in our study, plasma cortisol and lactate levels wereot well correlated, and even an opposite trend was observedor BNF and BaP. A similar result has been already observedy Pacheco and Santos (2001a). This antagonistic interactionf prolonged stress and PAH exposure was unexpected, anderhaps indicates that lactate levels are regulated in a differ-nt way than that of glucose by the interaction between effectsf toxicants and previous stress.

It is known that behavioural and physiological stressesponses in fish, like in other vertebrates, are linked by com-on control mechanisms in the brain (Øverli et al., 2005). The

rain monoamine neurotransmitters (mainly NA, DA and 5-HT)re believed to be involved in the control and integration ofhose behavioral and physiological stress responses in teleostsh (Øverli et al., 1999, 2001). It is known that cortisol release

s under the control of the HPI axis (Mommsen et al., 1999).CTH, the main secretagogue for cortisol is released from pitu-

tary, and this release is under the stimulatory control of theorticotropin-releasing factor (CRF) produced in the hypothala-us (Wendelaar Bonga, 1997; Fryer, 1989). At the same time,

ortisol may modulate ACTH release in fish hypothalamus andituitary (Mommsen et al., 1999). We have previously shown

hat PAHs (NAP and BaP) and PAH-like substances (BNF) may

odulate, in a non-specific manner, levels and turnovers of sero-onergic, dopaminergic and noradrenergic systems in the brainf rainbow trout (Gesto et al., 2006), but whether those changes

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re able to affect an endocrine response regulated by the centralervous system of the fish is not known.

A repeated effect of stress (in stressed-control fish withespect to unstressed fish) in brain monoamines was observedn our study, with increases in the content of DA and 5HT

etabolites (DOPAC and 5HIAA, respectively), in various brainegions (telencephalon, hypothalamus, preoptic region, braintem and optic tectum). The increase of metabolite levels wasften accompanied by increases in the metabolite/monoamineatios, used as a more direct index of activity. These data aren accordance with previous studies about stress effects in brain

onoamines of rainbow trout system (Øverli et al., 2001; Lepaget al., 2002). The effects of PAHs on the neurotransmitter contentfter stress stimuli were dependent on the hydrocarbon, brainegion or type of stress stimulus. A similar non-uniform pat-ern of changes had been previously observed for PAHs effectsn brain neurotransmitters in unstressed rainbow trout (Gestot al., 2006), and could be attributed to a non-specific interac-ion of those compounds that accumulate in lipophilic tissuesnd alter the normal function of membranes in nervous system.owever, the observed effects of PAHs on brain monoaminesf acutely or prolonged stressed fish were in general inhibitory,hus counteracting the stimulatory effects induced by the stres-ors. Changes in the levels of monoamines induced by PAHsended (mainly after prolonged stress) to approximate levels oftressed fish to those of unstressed ones, clearly counteractinghe effects of stress. This finding correlates well with the fall oflasma cortisol levels in fish treated with BNF and BaP afterrolonged stress, with respect to stressed-controls.

Interactions between different signalling systems involvedn the stress response is so complex that trying to distinguishetween causes and consequences may appear futile (Øverlit al., 2005). In this sense, in mammals it is known that bothopaminergic and serotonergic systems may be affected by cor-icosteroids (Chaouloff, 2000; Barrot et al., 2001) and observedAH effects on brain monoamines could be a consequence ofhanges in plasma cortisol levels and not its cause. Althoughhe present data does not allow to draw a direct link betweenlterations in brain amines and differences in the stress responsefter acute or prolonged stress, the results suggest that alterationsn brain neurotransmission processes might be involved in theAH-induced impairment of the endocrine responses to stress.

In conclusion, our results demonstrated that PAHs of lowNAP) and high (BaP) molecular weight, as well as PAH-ike substances known to activate AhR (BNF), can alter stressesponse of the fish in terms of neuroendocrine action, and lev-ls of plasma cortisol and metabolites. The responses observedaried with the duration of the stress stimuli, and particularlyfter a prolonged stress they address a reduction in fish abilityo cope with stressor, which is important for the assessment ofhe effects of fish populations in nature exposed to PAHs.

cknowledgements

This work was supported by grants VEM2003-20062 (Min-sterio de Ciencia y Tecnologıa and FEDER, Spain) andGIDT04PXIC31208PN (Xunta de Galicia, Spain), to J.L.

H

logy 86 (2008) 341–351

oengas and grant AGL2004-08137-c04-03/ACU (Ministerioe Educacion y Ciencia and FEDER, Spain) to J.M. Mıguez.anuel Gesto is recipient of a Xunta de Galicia predoctoral

ellowship.

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