The effects of polycyclic aromatic hydrocarbons on the immune system of fish: A review

Aquatic Toxicology 77 (2006) 229–238

Review

The effects of polycyclic aromatic hydrocarbons on the immune system of fish: A review S. Reynaud a,b,∗ , P. Deschaux b a

b

Laboratoire d’Ecologie Alpine. UMR CNRS 5553. Universit´e Joseph Fourier. BP 53. 38041 Grenoble cedex 9, France Laboratory of General and Comparative Immunophysiology, Science Teaching and Research Unit, 123, av. Albert Thomas, 87060 Limoges, France Received 21 February 2005; received in revised form 25 October 2005; accepted 25 October 2005

Abstract Polycyclic aromatic hydrocarbons are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. This review summarizes the diverse literature on the effects of these pollutants on innate and acquired immunity in fish and the mechanism of PAHinduced immunotoxicity. Among innate immune parameters, many authors have focused on macrophage activities in fish exposed to polycyclic aromatic hydrocarbons. Macrophage respiratory burst appears especially sensitive to polycyclic aromatic hydrocarbons. Among acquired immune parameters, lymphocyte proliferation appears highly sensitive to polycyclic aromatic hydrocarbon exposure. However, the effects of polycyclic aromatic hydrocarbons on both specific and non-specific immunity are contradictory and depend on the mode of exposure, the dose used or the species studied. In contrast to mammals, fewer studies have been done in fish to determine the mechanism of polycyclic aromatic hydrocarboninduced toxicity. This phenomenon seems to implicate different intracellular mechanisms such as metabolism by cytochrome P4501A, binding to the Ah-receptor, or increased intracellular calcium. Advances in basic knowledge of fish immunity should lead to improvements in monitoring fish health and predicting the impact of polycyclic aromatic hydrocarbons on fish populations, which is a fundamental ecotoxicological goal. © 2005 Elsevier B.V. All rights reserved. Keywords: Polycyclic aromatic hydrocarbons; Immunotoxicity; Fish

Contents 1. 2. 3.

4. 5. 6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of PAH on specific immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of PAH on non-specific immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Effects on lysozyme activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Effects on fish phagocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Effects on non-specific cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of PAH on fish resistance to pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PAHs induce fish immune system cell apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of PAH-induced immunotoxicity in fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Role of Ah-receptor or cytochrome P4501A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Role of intracellular calcium mobilization in PAH-induced immunotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: Ah-R, Ah receptor; a-NF, ␣-naphthoflavone; BaP, Benzo[a]pyrene; [Ca2+ ]i , intracellular calcium concentration; Con A, concanavalin A; ER, endoplasmic reticulum; DMBA, 7,12-dimethylbenz[a]anthracene; MAF, macrophage activating factor; MHC, Major histocompatibility complex; LPS, lipopolysaccharide; PAHs, polycyclic aromatic hydrocarbons; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PTK, protein tyrosine kinase; 3-MC, 3-methylcholanthrene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCR, T-cell receptor; XRE, xenobiotic response elements ∗ Corresponding author. Tel.: +33 04 76 51 46 80; fax: +33 04 76 51 44 63. E-mail address: [email protected] (S. Reynaud). 0166-445X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2005.10.018

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1. Introduction Polycyclic aromatic hydrocarbons (PAHs) have become widespread environmental contaminants due to their occurrence in petroleum, coal, soot, air pollutants and cutting oils (Hardin et al., 1992; Cooke and Denis, 1988; Mudzinski, 1993). PAHs have also contaminated both marine and freshwater systems (Couch and Harchbarger, 1985; Weeks et al., 1990). These compounds are immunotoxic, carcinogenic chemicals and the best studied are 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BaP) and 3-methylcholanthrene (3-MC) (White et al., 1985; White, 1986; Thurmond et al., 1987; Davila et al., 1995). BaP and 3-MC are found environmentally, whereas certain methylated PAHs, such as DMBA have been synthesized as model compounds (Krieger et al., 1995). Planar aromatic hydrocarbons trigger the induction of cytochrome P450 1A proteins via an intracellular aryl hydrocarbon receptor (Ah-R) (Poland and Knutson, 1982), predominantly found in liver but also in extrahepatic tissues (Fig. 1). The inactive AhR resides in the cytoplasm, in a complex with the molecular chaperone heat shock protein 90 (hsp90), the immunophilin XAP2 (also called AIP or ARA9), as well as the cochaperone p23 (Petrulis and Perdew, 2002; Backlund and Ingelman-Sundberg, 2005). Upon binding of a ligand, such as the high-affinity ligand tetrachlorodibenzo-pdioxin (TCDD), the receptor translocates to the nucleus where it

dimerizes with arnt (AhR nuclear translocator), which serves as a common dimerization partner for several bHLH-PAS proteins, including the AhR and hypoxia-inducible factor-1␣ (Petrulis and Perdew, 2002; Backlund and Ingelman-Sundberg, 2005). The AhR–arnt heterodimer binds to specific xenobiotic response elements (XREs) present in target genes, of which the best characterized, is the cytochrome P450 1A1 (CYP1A1) gene (Petrulis and Perdew, 2002; Backlund and Ingelman-Sundberg, 2005). In addition to this phenomenon, Ah-receptor ligands such as TCDD, 3-MC and related compounds enhance induction of phase II enzymes such as UDP glucuronosyltransferase (UDPGT) and glutathione S-transferase (GST) in mammals and fish (Goksøyr and Husøy, 1998; Taysse et al., 1998) (Fig. 1). The existence of Ah-R in fish and induction of P450 by planar hydrocarbons has been demonstrated (Taysse et al., 1998; Ferraris et al., 2005; Yamauchi et al., 2005). Moreover, the regulation of Ah-R mediated P450 induction by phosphorylation/dephosphorylation has been demonstrated in fish (Ferraris et al., 2005). Induction of P450 by PAHs has received the most attention as biomarkers and bioindicators of exposure (Huggett et al., 1992). PAHs, may also alter both specific and non-specific immunity in fish and mammals (White et al., 1994; Faisal and Huggett, 1993; Carlson et al., 2004a,b). There is now evidence from immunotoxicological risk assessment studies on rodents

Fig. 1. Mechanism of P4501A induction. Planar aromatic hydrocarbons trigger the induction of cytochrome P450 1A proteins via an intracellular aryl hydrocarbon receptor (Ah-R). AhR resides in the cytoplasm, in a complex with the molecular chaperone heat shock protein 90 (hsp90), the immunophilin XAP2 (also called AIP or ARA9), as well as the cochaperone p23. Upon binding of a PAH, the receptor translocates to the nucleus where it dimerizes with arnt (AhR nuclear translocator). The AhR–arnt heterodimer binds to specific xenobiotic response elements (XREs) present in target genes, of which the best characterized, is the cytochrome P450 1A1 (CYP1A1) gene. In addition to this phenomenon, Ah-receptor ligands enhance induction of phase II enzymes such as UDP glucuronosyltransferase (UDPGT) and glutathione S-transferase (GST) in mammals and fish (Goksøyr and Husøy, 1998; Taysse et al., 1998).

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and other mammals suggesting that xenobiotic-mediated suppression of innate immune responses may have more impact on resistance to pathogens than suppression of an acquired immune response (Luster et al., 1988). Fishes collected from PAH-polluted waters have external abnormalities thought to be related to immunosuppression, including lesions of gills, skin, and fins caused by opportunistic infections (Seeley and WeekPerkins, 1991). However, little is known about the mechanism by which PAHs induce immunotoxicity in fish. Recent studies in fish have investigated the mechanism of PAH-induced immunotoxicity (Faisal and Huggett, 1993; Reynaud et al., 2001, 2002, 2003, 2004; Carlson et al., 2004a,b; Reynaud and Deschaux, 2005). The intracellular mechanism implicated in PAH-mediated immunotoxicity in fish appeared to be similar to those observed in mammals and differed between species and PAHs studied (Faisal and Huggett, 1993; Reynaud et al., 2001, 2002, 2003, 2004; Carlson et al., 2004a,b; Reynaud and Deschaux, 2005). The purpose of this study was to synthesize recent findings concerning the effect of PAH on the fish immune system and the mechanism of PAH-induced immunotoxicity in fish. 2. Effects of PAH on specific immunity The acquired immune defense mechanisms of fish are the same as those for mammals and include cell- and humoralmediated responses (Zelikoff, 1998). As observed in the mammalian immune system, each of these specific sets of immune responses shows negative memory (tolerance), as well as positive anamnestic (memory) characteristics of a quicker, more prolonged immune response after a second contact with foreign antigen (Zelikoff, 1998). The major histocompatibility complex (MHC) and T-cell receptor (TCR) involved in specific recognition of antigens have been isolated from teleosts (Nakanishi, 2002; Rombout et al., 2005; Secombes et al., 2005). Immune cells producing antibodies, thought to be analogous to mammalian B-lymphocytes, are located in the spleen and kidney of fish (Zelikoff, 1998). These cells display surface immunoglobulin (Ig) and may be stimulated to proliferate by exposure to lipopolysaccharide (LPS) (Reynaud and Deschaux, 2005). However, unlike the five classes of Ig found in mammals, antibodies produced by fish appear to be restricted to two classes of Ig closely resembling mammalin IgM and IgD (Zelikoff, 1998; Srisapoome et al., 2004). Recently a novel class of antibody called IgT has been identified in rainbow trout (Oncorhynchus mykiss) (Hansen et al., 2005). Cell mediated immunity in teleosts is also comparable to that found in mammals. T-lymphocytes are the primary cell type responsible for cell-mediated immunity in fish (Secombes et al., 2005). T-lymphocytes from numerous species proliferate in response to the mitogens phytohemagglutinin (PHA) and concanavalin A (ConA) (Luebke et al., 1997; Reynaud et al., 2003). These cells also participate in other cell-mediated immune activities such as graft rejection (McKinney et al., 1981; Sarder et al., 2003), hypersensitivity reactions (Baldo and Fletcher, 1975; Nakanishi et al., 1999) responses to allogeneic cell stimulation (Miller et al., 1986), graft versus host rejection (Fisher et al.,

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2005) and elimination of virus-infected cells (Fisher et al., 2005). The classical division of T cells into cytotoxic and helper subpopulations is probably relevant in fish, based on these functional activities and MHC class I and II molecules, but as yet even this has not been definitively proven (Secombes et al., 2005; Fisher et al., 2005). In mammals, the effects of PAHs on B cell function are contradictory and dependent on the dose and the compound tested (Smialowicz et al., 1997). 3-MC and TCDD produce a marked depression in serum antibody titers in mice immunized with sheep erythrocytes (Davila et al., 1995; Smialowicz et al., 1997), whereas 2,2 ,4,4 ,5,5 -hexachlorobiphenyl enhances this production (Smialowicz et al., 1997). PAHs also suppress both the generation of cytotoxic T lymphocytes and T cell proliferation in response to mitogens (Thurmond et al., 1988; Davila et al., 1996). Previous studies on fish have also shown the same contradictory effect of PAHs on specific immunity as in mammals. TCDD suppresses antibody production against sheep red blood cells (SRBC) in Chinook salmon (Oncorhynchus tshawytscha) (Arkoosh et al., 1994). In tilapia (Oreochromis niloticus) BaP suppresses B cell-mediated immunity at 15 mg kg−1 whereas it increases this immunity at 25 mg kg−1 (Smith and Suthers, 1999). PAHs have been shown to suppress T-lymphocyte proliferation in spot (Leiostomus xanthurus) (Faisal and Huggett, 1993). Carlson et al. (2002) have shown that a single intraperitoneal injection of BaP produced a marked depression in lymphocyte proliferation in Japanese medaka (Oryzias latipes). In recent studies Carlson et al. (2004a,b) showed that BaP markedly depressed lymphocyte proliferation and antibody-forming cell numbers in Japanese medaka (Oryzias latipes). In carp (Cyprinus carpio), we have recently shown that T- and B-lymphocyte proliferation induced by ConA and LPS were inhibited by 3MC (0.5–50 ␮M) in vitro (Reynaud et al., 2003). In rainbow trout (Oncorhynchus mykiss), Tahir and Secombes (1995) have shown that intraperitoneal injection of diesel oil-based drilling mud extracts produced elevated head kidney lymphocyte proliferation in response to PHA. Resting lymphocyte proliferation following PAH exposure has not been documented in mammals or fish, but several studies in mammals clearly demonstrate a correlation between PAH exposure and the formation of DNA adducts in lymphocytes and macrophages (Johnsen et al., 1997; Rodriguez et al., 2002). We have recently demonstrated in carp that intraperitoneal injection of 3-methylcholanthrene (40 mg kg−1 ) induced a marked lymphocyte proliferation. The magnitude, sensitivity and specificity of 3-MC-induced lymphocyte proliferation suggest that this immune response may be the first immune biomarker of PAH exposure in fish (Reynaud et al., 2005). The capacity of 3-MC to induce lymphoproliferation appeared to be similar to the capacity of BaP to induce transient skin neoplasia in mice (Chakravarti et al., 2000). The underlying mechanism is unclear, but it was possible that, as in mouse skin treated with BaP, carp lymphocytes exposed to 3-MC underwent rapid clonal expansion following mutations. It has been shown in mouse skin that the mutations appeared starting at 1 day post-exposure in cells in phase S causing immediate proliferation (Chakravarti et al., 2000). More

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Lysozyme is an enzyme that disrupts bacterial cell walls by splitting glycosidic linkages in the peptidoglycan layers. It acts directly on the walls of Gram-positive bacteria, and on the inner peptidoglycan layers of Gram-negative bacteria, after complement and other enzymes have disrupted the outer walls (Yano et al., 1996). For toxicological studies with fish, lysozyme levels have been most frequently examined in plasma or serum. Laboratory, microcosm and field studies have been used to examine the impact of several complex toxic mixtures of environmental importance: diesel-oil drilling mud, and oil-contaminated sediments on lysozyme levels. When rainbow trout (Oncorhynchus mykiss) were injected with diesel-oil drilling mud, serum lysozyme levels were reduced at a dose of 0.6 ml kg−1 but not at higher doses (Tahir and Secombes, 1995). Experimental exposure of dab (Limanda limanda) to oil-contaminated sediments resulted in a decrease in serum lysozyme activity and serum lysozyme levels were reduced in dab caught near an oil-tanker accident (Secombes et al., 1997). However, dab exposed to sediment contaminated with PAH displayed no change in lysozyme activity (Hutchinson et al., 2003).

Fish collected from PAH-polluted waters have external abnormalities that are thought to be related to immunosuppression, including lesions of gills, skin and fins due to opportunistic infections (Seeley and Week-Perkins, 1991). Most authors have focused on macrophage activities in such fish and have reported decreases in phagocytosis and chemotaxis (Weeks and Warinner, 1984; Weeks et al., 1986; Seeley and WeekPerkins, 1991; Rice and Schlenk, 1995), whereas Kelly-Reay and Weeks-Perkins (1994) clearly demonstrated that killifish (Fundulus heteroclitus) chronically exposed to very high levels of PAHs showed increased-macrophage oxidative function. We have demonstrated that in vivo exposure of carp to 3-MC increases their macrophage respiratory burst activity (Reynaud et al., 2002). However, Carlson et al. (2004b) have recently shown that intraperitoneal injection of BaP depressed Japanese medaka phagocyte-mediated superoxide generation. In vitro exposure to 3-methylcholanthrene increases phorbol 12myristate 13-acetate (PMA)-induced respiratory burst activity in macrophages isolated from carp head kidney (Reynaud et al., 2001). By comparing the time course response of 3-MC on macrophage respiratory burst and biotransformation activity in carp, we demonstrated that macrophage oxidative function may be an equally sensitive marker of exposure to PAH as are biotransformation-related activities. This idea was highlighted many years ago since fish collected from polluted rivers displayed altered phagocytic cell function (Weeks and Warinner, 1984; Weeks et al., 1986; Seeley and Week-Perkins, 1991).

3.2. Effects on fish phagocytes

3.3. Effects on non-specific cytotoxicity

Phagocytosis is a primordial mechanism in all metazoan organisms. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens (Neumann et al., 2001). These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens (Neumann et al., 2001). In mammals, the mechanism of reactive oxygen species production is well understood (Fig. 2). However little is known about the biochemical structure of the enzymes involved in the respiratory burst of fish phagocytes. Secombes et al. (1992) demonstrated the presence of a b-type cytochrome (similar to gp91 and p21) that localized to the plasma membrane of fish phagocytes. Fish phagocyte respiratory burst can be activated in vitro by soluble mediators such as macrophage activating factor (MAF), produced by mitogen-activated kidney or peripheral blood leucocytes (Graham and Secombes, 1988; Duchiron et al., 2002a,b). Inducible nitric oxide (NO) production by fish macrophages has been a recent discovery (Schoor and Plum, 1994). Most recently, Yin et al. (1997) have demonstrated that MAF activated catfish (Ictalurus punctatus) macrophages produced nitric oxide. The molecule responsible for leukocyte supernatant-induced NO production in fish has been recently characterized as transferrin (Stafford et al., 2001).

The most extensively studied cytotoxic cells in teleosts are the non-specific cytotoxic cells (NCCs). Originally described in channel catfish, these cells are able to spontaneously kill a variety of xenogenic targets, including certain fish parasites and traditional mammalian NK cell targets (Graves et al., 1985; Evans et al., 1992; Shen et al., 2002). Unlike mammalian NK cells, catfish NCCs are small agranular lymphocytes that are commonly found in lymphoid tissues (i.e. pronephros and spleen), but rarely in the blood. These cells have been defined by reactivity with a monoclonal antibody (5C6), which reacts with a 32–34 kDa cell surface protein termed NCC receptor protein 1 (NCCRP-1) (Evans et al., 1988; Jaso-Friedmann et al., 1997; Shen et al., 2002). This receptor is believed to recognize a single, highly conserved target antigen (NKTag) found on potential targets ranging from protozoan parasites to human tumour cells. NCC-like activity has been shown in other fish species, including rainbow trout (Oncorhynchus mykiss) (Greenlee et al., 1991), carp (Cyprinus carpio) (Suzumura et al., 1994), damselfish (Abudefduf sordidus) (McKinney and Schmale, 1994), and tilapia (Oreochromis aureus) (Faisal et al., 1989). Because of the ability of fish NCCs to kill traditional mammalian NK targets, NCCs have been postulated to be the evolutionary precursor of mammalian NK cells (Graves et al., 1985; Evans et al., 1988; Jaso-Friedmann et al., 1997; Shen et al., 2002). NCC activity appears to be sensitive to phenols and PAHs. In carp, hydroquinone reduced NCC activity both in vivo and in vitro, but phenol and pyrocatechol decreased NCC activity

studies are needed both in fish and mammals to explain this phenomenon. 3. Effects of PAH on non-specific immunity 3.1. Effects on lysozyme activity

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Fig. 2. Assembly of respiratory burst oxidase within the membrane of vertebrate phagocytes. Phosphorylation of cytosolic components (p47-phox and p67-phox) by protein kinases (PKC, protein kinase C, PKA, protein kinase A, MAPK, mitogen activated protein kinases) initiates translocation of the cytosolic complex to the cell membrane. At the membrane, these cytosolic components interact with membrane bound cytochrome (gp91-phox and p21-phox), and in the presence of GTP-rac proteins, form the functional respiratory burst oxidase. Activity of this multi component enzyme is regulated by rho proteins and by phosphatases that regulate levels of GTP/GDP binding and phosphorylation of cytosolic components (modified from Neumann et al., 2001).

only in vitro (Taysse et al., 1998). Perhaps, the most significant NCC response has been seen in oyster toadfish (Opsanus tau) after intraperitoneal injection of DMBA, which was used as a representative PAH. NCC activity was nearly abolished at all DMBA doses (Seeley and Week-Perkins, 1997) and appeared more vulnerable to impairment than macrophage phagocytosis. NCC activity has also been measured in fish from different field sites. NCC activity of mummichog (Fundulus heteroclitus) from the Elizabeth River, which was heavily contaminated with PAHs, was significantly depressed relative to NCC activity of fish from the York River, which was much less contaminated (Faisal et al., 1991). By contrast, when NCC activity in the anterior kidney and spleen was compared in sunfish near a point-source of contamination to sunfish (Lepomis macrochirus) from sites further down-stream, NCC activities did not follow gradient–response patterns, as did phagocyte oxidative burst (Rice et al., 1996). Although laboratory and field studies so far suggest that NCC activity is not particularly sensitive to ecotoxicants in general, the development of better and more standard methods of evaluating NCC activity should improve the value of this cellular endpoint (Bols et al., 2001).

4. Effects of PAH on fish resistance to pathogens Carlson et al. (2002) have demonstrated that a single administration of BaP at 20 or 200 ␮g g−1 BW significantly increased host susceptibility to infection with the bacterial fish pathogen Yersinia ruckeri. Although BaP has not been shown previously in fish or mammals to reduce host resistance against infection, laboratory exposure of chinook salmon (O. tshawytscha) to another PAH, DMBA, decreased host resistance against subsequent challenge with the marine bacterial pathogen Vibrio anguillarium (Arkoosh et al., 1998). Effects upon medaka host resistance appears to conflict with findings in mammals which demonstrated that exposure to BaP had no effect upon the resistance of exposed mice against infection with Listeria monocytogenes (Dean et al., 1983). The discrepancy between results observed in medaka and those in mice may be due to differences in bacterial pathogenesis. While L. monocytogenes infection appears to rely primarily upon evasion of cell-mediated immunity (Bradley, 1995), a compartment of the immune system not highly sensitive to BaP-induced immunotoxicity (Dean et al., 1983); humoral immune defense mechanisms appear to play an important role

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in mediating Yersinia infection by producing antibodies against Y. ruckeri necessary for the opsonization and ultimate phagocytosis of the bacterium (Griffin, 1983; Siwicki and Dunier, 1993). Recently, Palm et al. (2003) have shown that juvenile Chinook salmon (Oncorhynchus tschawytscha) exposed to a PAH mixture over a period of 28 days did not display a decrease in host resistance to Listonella anguillarum. 5. PAHs induce fish immune system cell apoptosis Apoptosis is a programmed cell death process (Kerr et al., 1972) that plays a critical role in immunity from the development of the T cell repertoire in the thymus (Gillette-Ferguson and Sidman, 1994) to B and T cell peripheral tolerance (Ucker et al., 1992; Kamesaki et al., 1994; Yamaguchi et al., 1996). Weyts et al. (1997) have shown that apoptosis as an immune regulatory mechanism is conserved in fish demonstrating its importance in maintaining immunological homeostasis. Hence, factors that modulate programmed cell death have the potential to disrupt and/or to compromise lymphocyte repertoire development and immune responsiveness in both fish and mammals. In mammals, one part of immunotoxicity may be mediated by the induction of programmed cell death in lymphocytes (Burchiel et al., 1992; Hardin et al., 1992; Hinoshita et al., 1992; Yamaguchi et al., 1996). In fish, fewer studies have been done to define the role of apoptosis in PAH-mediated immunotoxicity. PAH induced apoptosis has been observed in fish. In vivo exposure to PAH induced apoptosis in eel erythrocytes (Nigro et al., 2002) and channel catfish (Ictalurus punctatus) ovary cells (Weber and Janz, 2001). Eelpout (Zoarces viviparous) living in PAH contaminated water displayed increased erythrocyte apoptosis (Frenzilli et al., 2004). Using three criteria (localisation of phosphatidylserine on the outer side of the cell membrane, chromatin condensation and fragmentation, and decreased cell size) we have recently shown that 3-MC dose dependently induces apoptosis in both lymphocytes and phagocytes (Reynaud et al., 2004). 6. Mechanism of PAH-induced immunotoxicity in fish The mechanism of PAH-induced immunotoxicity has been well studied in mammals. Several authors have suggested that metabolic activation is required for PAHs to produce immunosuppression (Mudzinski, 1993). This phenomenon has been described for 3-MC in murine cytotoxic T-lymphocytes (Davila et al., 1995). Others have shown that PAHs directly modulate Ca2+ homeostasis (Archuleta et al., 1993; Krieger et al., 1994; Mounho and Burchiel, 1998). Few studies in fish have examined the mechanism of PAH-induced immunotoxicity. 6.1. Role of Ah-receptor or cytochrome P4501A Faisal and Huggett (1993) have shown that PAHs suppress proliferative responses of lymphocytes in spot (Leiostomus xanthurus) and that this modulation may involve cytochrome P450-mediated PAH metabolism. Previous studies in our laboratory have shown that incubating carp adherent phagocytes

with increasing concentrations of 3-MC potentiates the production of ROS by cells stimulated with PMA, but it has no effect on unstimulated macrophages. However Ah-R and the cytochrome P450 1A inhibitor, ␣-naphthoflavone (␣-NF), reversed 3-MC potentiation of PMA-induced macrophage respiratory burst suggesting that metabolic activation of 3-MC by cytochrome P450 1A is responsible for the effects observed (Reynaud et al., 2001). Intraperitoneal 3-MC injection (40 mg kg−1 ) produced a marked increase in carp head kidney phagocyte ROS production which was inhibited by a-NF (Reynaud et al., 2002). Carlson et al. (2002) showed, in Japanese medaka (Oryzias latipes), that low doses of BaP inhibited B- and T-cell proliferation in the absence of elevated CYP 1A expression/activity suggesting that metabolic activation was not required to produce immunosuppression in fish. However, more recently, Carlson et al. (2004a,b) have shown that BaP-reduced lymphocyte proliferation, phagocyte-mediated superoxide generation and antibodyforming cell numbers in Japanese medaka was directly dependent on the production of BaP metabolites in lymphoid tissues. In mammals, the role of PAH metabolism or PAH binding to AhR in immunotoxicity is unclear (Davila et al., 1996). The capacity of a-NF to inhibit PAH-induced immunotoxicity has been observed in mammals with TCDD-induced suppression of humoral immunity (Blank et al., 1987) and T-cell activity (Davila et al., 1995). Since a-NF also inhibits cytochrome P450 isoenzymes (Goujon et al., 1972; White et al., 1994), several authors have suggested that metabolic activation by cytochrome P450 1A enzymes may be responsible for some of the immunotoxic effects of BaP (Kawabata and White, 1987), DMBA (Ladics et al., 1991) and 3-MC (Davila et al., 1995) in mammals. Binding to AhR is an essential step for halogenated aromatic hydrocarbon mediated immunotoxicity (Holsapple et al., 1991). DMBA has been shown to inhibit B- and T-cell activity in a metabolism independent manner (Thurmond et al., 1987, 1988; Davila et al., 1995). A combination of metabolic activation and direct effects of BaP has been shown to be responsible for its immunotoxic effects (Mudzinski, 1993; Davila et al., 1995). Taken together these studies suggested that mechanisms involved in PAH-induced immunosuppression have been phylogenetically conserved from fish to mammals. 6.2. Role of intracellular calcium mobilization in PAH-induced immunotoxicity In order to explain the effects of 3-MC on both specific and non specific immunity, we suggested its capacity to modulate intracellular calcium levels [Ca2+ ]i . For the first time in fish, we found that the 3-MC dose dependently increased [Ca2+ ]i in phagocytes and lymphocytes from carp (Reynaud et al., 2001, 2003). This dose dependence has been observed for BaP (Tannheimer et al., 1999) and DMBA (Burchiel et al., 1991) in mammals. We observed that this increase in [Ca2+ ]i was rapid; and as rapid as that described for BaP in mammalian models (Krieger et al., 1994). A kinetic study of 3-MC-induced calcium mobilization has shown that this hydrocarbon produced a sustained increase in [Ca2+ ]i (2 h minimum) both in phagocytes and lymphocytes (Reynaud et al., 2001, 2003). This phenomenon

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has been previously observed in mammals and correlated with the immunosuppressive properties of BaP (Krieger et al., 1994), DMBA (Burchiel et al., 1991) and TCDD (Karras et al., 1995). As for 3-MC, a-NF produced a dose- and time-dependant elevation of calcium levels in carp phagocytes and lymphocytes (Reynaud et al., 2001, 2003). The fact that a-NF produced a marked elevation in [Ca2+ ]i but was not able to increase PMAinduced ROS production suggests that other mechanisms, in addition to calcium mobilization, could be responsible for ROS production (Reynaud et al., 2001). In contrast, the fact that both 3-MC and a-NF inhibited lymphocyte proliferation and induced calcium mobilization suggests that 3-MC-inhibited B- and Tcell proliferation is calcium dependent (Reynaud et al., 2003; Reynaud and Deschaux, 2005). However, in carp we have found that 3-MC induced-apoptosis is calcium dependent in both lymphocytes and phagocytes (Reynaud et al., 2004). The biochemical and cellular physiological cascade of events involved in B- and T-lymphocyte activation is, for the most part, well understood. Membrane events, including PTK (G protein) and phospholipase activation, intracellular calcium mobilization and PKC activation are involved (Davila et al., 1995). The activation of neutrophils and macrophages leading to increased ROS production is also for the most part, well known. Membrane events including protein tyrosine kinase (G protein) and phospholipid lipase activation, intracellular calcium mobilization, PKC activation and activation of oxidase are also involved (Rice et al., 1996). However, in carp phagocytes and lymphocytes 3-MC did not induce PTK activation suggesting that G proteins were not implicated in 3-MC-induced calcium mobilization (Reynaud et al., 2001, 2003). Furthermore, in these cells the capacity of 3-MC to induce calcium mobilisation was independent of Ca2+ influx from extracellular calcium stores but was dependent on calcium influx from the endoplasmic reticulum (Reynaud et al., 2001, 2003). Pharmacological studies in carp phagocytes and lymphocytes have revealed the capacity of 3MC to directly inhibit endoplasmic reticulum calcium ATPases (Reynaud et al., 2001, 2003, 2004). 7. Conclusion The immune system of fish is very sensitive to PAH pollution; and these pollutants affect both non-specific and specific immunity. However, the effects observed depend on the type of PAH, the route of administration, the concentration used and the species studied. The capacity of PAHs to induce immunotoxicity implies different intracellular mechanisms, such as PAH metabolism by cytochrome P450, binding to Ah-receptor and intracellular calcium mobilization. Comparative studies clearly demonstrate that these mechanisms of toxicity have been phylogenetically conserved from fish to mammals. References Archuleta, M.M., Schieven, G.L., Ledbetter, J.A., Deanin, G.G., Burchiel, S.W., 1993. 7,12-Dimethylbenz[a]anthracene activates protein-tyrosine kinases Fyn and Lck in the HPB-ALL human T-cell line and increases tyrosine phosphorylation of phospholipase C-gamma 1, formation of inos-

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