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Peroxisome Proliferation as a Biomarker in EnvironmentalPollution AssessmentMIREN P. CAJARAVILLE,* IBON CANCIO, ARANTZA IBABE, AND AMAIA ORBEABiologia Zelularra eta Histologia Laborategia, Zoologia eta Animali Zelulen Dinamika Saila, Zientzi Fakultatea, Euskal HerrikoUnibertsitatea, 644 P.K., E-48080 Bilbo, Basque Country, Spain

KEY WORDS peroxisomes; acyl-CoA oxidase; peroxisomal volume density; peroxisome prolif-erator-activated receptors; cellular biomarkers; aquatic organisms; pollution bio-monitoring

ABSTRACT Peroxisome proliferators comprise a heterogeneous group of compounds known fortheir ability to cause massive proliferation of peroxisomes and liver carcinogenesis in rodents. In recentyears it has become evident that other animals may be threatened by peroxisome proliferators, inparticular aquatic organisms living in coastal and estuarine areas. These animals are exposed to avariety of pollutants of industrial, agricultural and urban origin which are potential peroxisomeproliferators. Both laboratory and field studies have shown that phthalate ester plasticizers, PAHs andoil derivatives, PCBs, certain pesticides, bleached kraft pulp and paper mill effluents, alkylphenols andestrogens provoke peroxisome proliferation in different fish or bivalve mollusc species. The responseappears to be mediated by peroxisome-proliferator activated receptors, members of the nuclear receptorfamily, recently cloned in fish. Based on these results it is proposed that peroxisome proliferation couldbe used as a biomarker of exposure to a variety of pollutants in environmental pollution assessment.This is illustrated by a case study in which mussels, used worldwide as sentinels of environmentalpollution, were transplanted from reference to contaminated areas and vice versa. In mussels native toan area polluted with PAHs and PCBs, peroxisomal acyl-CoA oxidase (AOX) activity and peroxisomalvolume density were 2-3 fold and 5-fold higher, respectively, compared to the reference site. Whenanimals were transplanted to the polluted station, with increased concentration of organic xenobiotics,a concomitant significant increase of AOX was recorded. Conversely, in animals transplanted to thecleaner station, AOX activity and peroxisomal volume density decreased significantly. These resultsindicate that peroxisome proliferation is a rapid (i.e., two days) and reversible response to pollution inmussels. Before peroxisome proliferation can be implemented as a biomarker in biomonitoring pro-grams, a well-defined protocol should be established and validated in intercalibration and qualityassurance programmes. Furthermore, the influence of biotic and abiotic factors, some of which areknown to affect peroxisome proliferation (season, tide level, interpopulation and interindividual vari-ability), should be taken into consideration. The possible hepatocarcinogenic effects as well as thepotential adverse effects on reproduction, development, and growth of peroxisome proliferators areunknown in aquatic organisms, thus providing a challenge for future investigations. Microsc. Res. Tech.61:000–000, 2003. © 2003 Wiley-Liss, Inc.

INTRODUCTIONPeroxisomes are ubiquitous single-membrane lim-

ited versatile organelles essential for �-oxidation offatty acids, especially very long-chain fatty acids. Theyare also important for many other cellular processesrelated to lipid metabolism, oxyradical homeostasis,catabolism of purines and polyamines, and metabolismof amino acids and glyoxylate (Cancio and Cajaraville,2000; Reddy and Mannaerts, 1994; Singh, 1997). As thelast organelle to be discovered (De Duve, 1965), itsroles in cell metabolism and function have been largelyunknown for years and its importance underestimated.Peroxisomes acquired relevance with the discoverythat fibrates, used as hypolipidemic drugs to lower thelipid levels in serum of patients with hypercholesterol-emia, caused in rodents a massive proliferation of per-oxisomes (Hess et al., 1965) and liver carcinogenesis(Reddy et al., 1980; Reddy and Lalwani, 1983). Theincrease in the volume and number of peroxisomes, a

Grant sponsor: CICYT, Grant sponsor: BEEP (European Commission, SpanishMinistry of Science and Technology; Grant number: AMB99-0324; ResearchDirectorate General, Environment Programme-Marine Ecosystems); Grantnumber: EVK3-CT2000-00025).

Abbreviations used: AOX, Acyl-CoA oxidase; B(�)P, Benzo(�)pyrene; BKME,bleached kraft pulp and paper mill effluent; COUP-TF, chicken ovalbumin up-stream promoter-transcription factor; DAB, 3,3�-diaminobenzidine; DDT, dichlo-rodiphenyl trichloroethane; DEHP, diethylhexyl phthalate; DNOC, dinitro-o-cresol; FTZ-F1, Fushi tarazu transcription factor-1; PAH POLYCYCLIC ARO-MATIC HYDROCARBON PCB, polychlorinated biphenyl; PH, peroxisomalhydratase-dehydrogenase-isomerase or multifunctional enzyme; PMP-70, perox-isomal membrane protein of 70 kD; PPAR, peroxisome proliferator-activatedreceptor; PPRE, peroxisome proliferator response element; RXR, retinoid Xreceptor; SKL, serine-lysine-leucine; VVP, peroxisomal volume density; WAF,water accommodated fraction.

*Correspondence to: Professor M.P. Cajaraville, Biologia Zelularra eta Histo-logia Laborategia, Zoologia eta Animali Zelulen Dinamika Saila, Zientzi Fakul-tatea, Euskal Herriko Unibertsitatea, 644 P.K., E-48080 Bilbo, Basque Country,Spain. E-mail: [email protected]

Received 14 September 2001; accepted in revised form 27 December 2001DOI 10.1002/jemt.10329Published online in Wiley InterScience (www.interscience.wiley.com).

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phenomenon known as peroxisome proliferation, isusually accompanied by induction of all three enzymesof the peroxisomal �-oxidation pathway, namely, acyl-CoA oxidase (AOX), peroxisomal hydratase-dehydroge-nase-isomerase or multifunctional enzyme (PH), andthiolase (Lazarow and De Duve, 1976; Reddy and Man-naerts, 1994). Enzyme induction occurs through tran-scriptional activation of the corresponding genes byperoxisome proliferator-activated receptors (PPARs),members of the nuclear receptor family (Issemann andGreen, 1990). PPAR forms a heterodimer with anothernuclear receptor, the retinoid X receptor (RXR), andthen the heterodimer binds to a specific DNA regula-tory element (peroxisome proliferator response ele-ment or PPRE) located in the promoter region of targetgenes. Gene transcription is activated in response tobinding of the ligand of either receptor (Kersten et al.,2000). Activating ligands for PPARs comprise both en-dogenous compounds such as fatty acids and eico-sanoids, and xenobiotics such as fibrate hypolipidemicdrugs (Forman et al., 1997; Kersten et al., 2000;Schoonjans et al., 1996).

In addition to fibrates and other hypolipidemicdrugs, peroxisome proliferators comprise a heteroge-neous group of compounds including phthalate esterplasticizers, chlorophenoxyacetate herbicides andother pesticides, steroids, solvents and diverse indus-trial chemicals, food derivatives, polycyclic aromatichydrocarbons (PAHs), and polychlorinated biphenyls(PCBs) (Beier and Fahimi, 1991; Bentley et al., 1993;Fahimi and Cajaraville, 1995; Lake, 1995). One com-mon feature of these compounds or their metabolicderivatives is a hydrophobic-lipophilic backbone withan acidic function, generally a carboxylic group (Fa-himi and Cajaraville, 1995). Long-term treatment withperoxisome proliferators causes development of hepa-tocarcinomas in rodents (Lake, 1995; Reddy et al.,1980; Reddy and Lalwani, 1983; Stott, 1988). As per-oxisome proliferators are non-genotoxic and non-muta-genic compounds, their carcinogenicity has been as-cribed to a number of different mechanisms includingincreased oxidative stress and DNA damage, increasedDNA synthesis and mitogenesis, promotion of initiatedliver foci, and suppression of apoptosis (Cancio andCajaraville, 2000; James and Roberts, 1995; Yeldandiet al., 2000). There is a strong correlation betweenperoxisome proliferation and hepatocarcinogenesis inrodents although a causal link remains to be estab-lished.

As humans are refractory to peroxisome prolifera-tion, this response has been seen as a rodent-specificphenomenon, with little importance for human health(Bentley et al., 1993; Doull et al., 1999; Roberts, 1999).In recent years, it has become evident that wildlife maybe threatened by peroxisome proliferators, thus lead-ing to a burgeoning of peroxisome research in the fieldof environmental toxicology. Aquatic animals living incoastal and estuarine areas are exposed to a variety ofpollutants, coming from industrial, agricultural, andurban contamination, which are potential peroxisomeproliferators. In this review, we give an overview ofevidence indicating that peroxisome proliferation oc-curs in aquatic organisms from polluted environments.Moreover, it is proposed that peroxisome proliferationcould be used as a biomarker of exposure to a variety of

pollutants in environmental pollution assessment.This is illustrated by a case study in which musselswere transplanted from reference to contaminated ar-eas and vice versa. Finally, different techniques avail-able for measurement of peroxisome proliferation inaquatic organisms are discussed and their advantagesand disadvantages highlighted.

PEROXISOME PROLIFERATION ANDPPARs IN AQUATIC ORGANISMS

Aquatic organisms are exposed to a variety of con-taminants in their environment. Some of these areknown for their ability to cause peroxisome prolifera-tion in rodent models. These include phthalate esterplasticizers, PAHs and oil derivatives, PCBs, and cer-tain pesticides. As reviewed recently (Cancio and Ca-jaraville, 2000; Fahimi and Cajaraville, 1995), increas-ing evidence suggests that aquatic organisms such asmolluscs and fish are responsive to these environmen-tal pollutants.

A decade ago, electron microscopic studies suggestedthat organelles identified morphologically as peroxi-somes proliferate in the kidney of the gastropod mol-lusc Littorina littorea exposed to the PAH naphthalene(Cajaraville et al., 1990a). Later research focused inmussels (Mytilus sp) because these filter-feeding bi-valve molluscs are used worldwide as a bioindicatorand sentinel species of environmental pollution in ma-rine and estuarine areas (Bayne, 1989; Goldberg,1986). Peroxisomes in the digestive gland of musselsshare several characteristics with vertebrate liver per-oxisomes but they also show some special features.They contain the marker enzyme catalase and enzymesinvolved in �-oxidation of fatty acids as well as theperoxisomal membrane protein PMP70 (Cajaraville etal., 1992; Cancio et al., 2000a), but apparently lack theenzymes involved in purine metabolism xanthine oxi-dase and urate oxidase (Cancio and Cajaraville, 1997,1999; Cancio et al., 2000b). In addition, molluscan per-oxisomes, and most evidently fish liver peroxisomeshost a variety of antioxidant enzymes, active in thescavenging of potentially harmful oxygen free radicals(Orbea et al., 2000; Pedrajas et al., 1996).

In mussels exposed for up to 91 days to three doses ofthe water accommodated fraction (WAF) of two crudeoils and one lubricant oil, peroxisome proliferation wasdetected in the first sampling (after 21 days), and theresponse disappeared afterwards (Cajaraville et al.,1997; Fahimi and Cajaraville, 1995). For mussels ex-posed to the Maya type crude oil, the heaviest oiltested, only those treated with the highest dose (40%)showed significant increases of peroxisomal volumedensity (Vvp). In the case of mussels treated with thelighter Ural type oil, groups exposed to the low andhigh doses (0.6 and 40%, respectively) showed a signif-icant 2–3-fold increase in this parameter. The mostmarked response was observed in mussels exposed tothe lubricant oil, with up to 5-fold increases in Vvp (Fig.1). The peroxisome proliferating ability of lubricant oilWAF in mussels was confirmed in another laboratoryexperiment (Cancio et al., 1998) in which the effects oflubricant oil WAF and the model PAH benzo(�)pyrene(B(�)P) were compared to those of typical mammalianperoxisome proliferators, i.e., the hypolipidemic drugclofibrate and the phthalate ester most widely used as

2 M.P. CAJARAVILLE ET AL.

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a plasticizer, diethylhexyl phthalate (DEHP). A signif-icant increase in AOX activity was recorded after7 days treatment, by injection every two days, withclofibrate, B(�)P, and DEHP, and 21 days of water-borne exposure with the lubricant oil WAF. Peroxiso-mal volume and numerical densities were also signifi-cantly increased in mussels injected with lubricant oilWAF or clofibrate. Similarly, increases in peroxisomeswere detected in mussels exposed to microencapsulatedPAHs (Krishnakumar et al., 1997). In a recent fieldstudy, increases in Vvp were recorded in mussels con-comitant to increased bioavailability of PAHs (Orbea etal., 1999b). Krishnakumar et al. (1995) found a positivecorrelation between the concentration of organic pol-lutants (PAHs, PCBs, DDTs) accumulated by musselsand peroxisome abundance in their digestive glandswhen a polluted and a reference station were comparedin the western United States coast. In a field studyalong the western Mediterranean coast, increased Vvpwas detected in mussels from Barcelona, the site show-ing highest levels of PAHs and PCBs in comparisonwith the remainder of the test sites (Porte et al., 2001).

In order to investigate if pollutants other than or-ganic xenobiotics were able to cause peroxisome prolif-eration in mussels, an experiment was conducted withthe heavy metal Cd alone or in combination with B(�)P(Orbea et al., 2002a). In mussels exposed to Cd for up to21 days, no increases in AOX or Vvp were detected,indicating that Cd does not cause peroxisome prolifer-ation in mussels. In the same experiment, musselsexposed to B(�)P alone or in mixture with Cd and toDEHP showed increased AOX activity after 21 days oftreatment. Hence, peroxisome proliferation appears tobe a specific response to certain organic contaminants(Table 1) but further studies with other metals shouldbe carried out to confirm this idea.

In fish, there are data indicating that fibrate hypo-lipidemic drugs, various pesticides, bleached kraft pulp

and paper mill effluents (BKMEs), PAHs, phthalates,alkylphenols, and estrogens can cause peroxisome pro-liferation (Table 1). Intraperitoneal injection of the hy-polipidemic drugs ciprofibrate and gemfibrozil pro-vokes increased levels of AOX, PH, and catalase, andincreased Vvp and liver-to-body weight ratios in rain-bow trout Oncorhynchus mykiss (Scarano et al., 1994;Yang et al., 1990). The Japanese medaka (Oryzias lati-pes) responds similarly to gemfibrozil exposure withincreases in peroxisomal AOX and PH (Scarano et al.,1994). Clofibrate or bezafibrate administration tosalmon (Salmo salar) hepatocytes in culture also re-sults in an increased AOX activity (Ruyter et al., 1997).Injection of the herbicide dieldrin in Sparus auratamarkedly induces the activity of AOX and protein con-centration of the peroxisomal fraction (Pedrajas et al.,1996). Dinitro-o-cresol (DNOC) exposure causes a stim-ulation of peroxisomal enzymes (catalase, allantoinaseand urate oxidase) and a higher number of peroxisomesin liver of the European eel Anguilla anguilla (Braun-beck and Volkl, 1991). Combined exposure to endosul-fan and disulfoton also provokes a transient increase inthe absolute volume occupied by peroxisomes in liver ofrainbow trout (Arnold et al., 1995) and peroxisomeproliferation has been reported in kidney proximal tu-bules of rainbow trout treated with atrazine or linuron(Oulmi et al., 1995a,b). BKMEs provoke increases incatalase, lauroyl-CoA oxidase, and AOX in the channelcatfish Ictalarus punctatus (Mather-Mihaich and DiGiulio, 1991) and cause an increase in the number ofliver peroxisomes in Cottus gobio down-stream of twopaper mills discharging BKME (Bucher et al., 1992).Intraperitoneal injection of B(�)P in the demersal fishSolea ovata results in increased numerical densities ofhepatic peroxisomes (Au et al., 1999). In zebrafishDanio rerio, exposure to the organochlorine pesticidemethoxychlor, the phthalate dibutylphthalate, the al-kylphenol 4-tert-octylphenol, and estrogens causes an

Fig. 1. Light micrographs of mussel digestive gland cryostat sec-tions stained with the DAB technique at alkaline pH to visualizeperoxisomes. A: Control mussel sampled at day 21. B: Mussel treatedwith the WAF of a lubricant oil for 21 days. In mussels treated with

lubricant oil, a massive peroxisome proliferation occurs. Peroxisomesreactive for DAB are indicated by arrows. Asterisks indicate hemo-cytes containing lipofuscin granules unspecifically stained with DAB.Scale bars � 20 �m.

3PEROXISOME PROLIFERATION AS POLLUTION BIOMARKER

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increase in liver peroxisomal volume, surface, and nu-merical densities together with significant inductionsof AOX activity (Ortiz-Zarragoitia and Cajaraville,2000). A slight peroxisome proliferation in liver wasobserved after injection of 4-chloroaniline in early lifestages of zebrafish (Oulmi and Braunbeck, 1996).

Although still fragmentary and related to few speciesof aquatic organisms, the data summarized above sug-gest the occurrence of peroxisome proliferation in non-mammalian organisms. Peroxisome proliferation hasbeen reported in mussel digestive gland and fish liverbut, most significantly, fish kidney appears to be re-sponsive too (Oulmi et al., 1995a). The mechanismunderlying peroxisome proliferation in mammals in-volves “peroxisome proliferator activated receptors”(PPARs) belonging to the nuclear receptor family (Is-semann and Green, 1990; Schoonjans et al., 1996). Todate, three subtypes have been described: PPAR�,PPAR�, and PPAR�. PPAR� is the subtype involved inperoxisome proliferation and activates genes involvedin lipid catabolism (Schoonjans et al., 1996). PPAR�appears to play diverse roles in basic lipid metabolismand participates in embryo implantation and develop-ment whereas PPAR� has a key function in the differ-entiation of adipocytes, being mostly involved in adipo-genesis (Kersten et al., 2000; Peters et al., 2000). Sev-eral recent reports have also involved PPAR� inaddition to PPAR� in the activation of the peroxisomeproliferation response (Aperlo et al., 1995; Ma et al.,1998).

PPARs have recently been found in the Atlanticsalmon S. salar (Andersen et al., 2000; Ruyter et al.,1997) and the plaice Pleuronectes platessa (Leaver etal., 1998). In both cases, a PPAR form has been clonedand found to be similar to PPAR� (overall amino acidsequence identity of 43–48% of salmon PPAR to Xeno-pus or mammalian PPAR�, Andersen et al., 2000). Inzebrafish, 100 bp of the DNA-binding domain of each ofthe three subtypes has been cloned (Escriva et al.,1997). We have recently detected expression of the

three subtypes in several tissues of adult zebrafish byimmunohistochemistry (Ibabe et al., submitted—b).PPAR� was expressed mainly in liver hepatocytes,proximal tubules of kidney, enterocytes, and pancreas(Fig. 2). This is in agreement with PPAR� distributionin mammals and amphibia predominantly in tissuesthat catabolize high amounts of fatty acids (Braissantet al., 1996; Dreyer et al., 1992; Lemberger et al.,1996a). PPAR� showed a widespread distribution inzebrafish whereas PPAR� was expressed weakly in fewcell types (Ibabe et al., submitted-b). Further studiesare needed to decipher the function(s) of PPAR sub-types in zebrafish and other fish species. Remarkably,there is one report indicating induction of PPAR� tran-scription in salmon hepatocytes by clofibrate and beza-fibrate (Ruyter et al., 1997). With regard to inverte-brates, to the best of our knowledge no information isavailable as to the possible existence of PPARs in mol-luscs or other invertebrates (Stewart et al., 1994). Es-criva and coworkers (1997) extensively searched fornuclear receptors in cnidarians and platyhelminthesand they were only able to identify members of theCOUP-TF, RXR, and FTZ-F1 groups of receptors, butnone from any other group including PPAR.

Peroxisome proliferation is a highly species-specificprocess occurring in many rodents but not in severalother mammalian organisms including guinea pigs,humans, and non-human primates (Fahimi et al., 1993;Pacot et al., 1996). Similarly, peroxisome proliferatorselicit liver carcinogenesis only in responsive organismssuch as rodents but not in humans. The possible occur-rence of carcinogenesis in response to peroxisome pro-liferators in fish, mussels, or other responsive aquaticorganisms is unknown. Quantitative and qualitativedifferences in PPAR� expression appear to hold the keyto species differences in the effects of peroxisome pro-liferators (Holden and Tugwood, 1999; Roberts, 1999).Interestingly, induction of peroxisomal �-oxidation en-zymes and PPAR occur in salmon or rainbow trouthepatocytes treated with clofibrate (Donohue et al.,

TABLE 1. Main features of peroxisome proliferation as biomarker of organic pollution accordingto studies carried out in molluscs (M) or fish (F)*

Feature References

Specificity Fibrate hypolipidemic drugs (M, F) (8, 10, 24, 26, 27)Phthalates (M, F) (8, 17, 19)PAHs (M, F) (2, 5, 6, 7, 8, 13, 16, 17)PCBs (M) (13)BKMEs (F) (4, 14)Alkylphenols (F) (19)Estrogens (F) (19)Various pesticides (F) (1, 19, 20, 21, 22, 23)

Influence of biotic factors Limited knowledge on influence of gender (M, F),developmental stage (F) and age (F)

(11, 12, 15, 25)

Influence of abiotic factors Limited knowledge on seasonal variations (M, F) (9, 15, 16, 18, 25)Reversibility Proven in transplant experiment (M) but more

knowledge neededThis report

Repeatability Shown in experiments with B(a)P and DEHP (M) (8 and 17)Reproducibility Interlaboratory studies neededEcological relevance (effects on reproduction,

development or growth)Not known

*PAHs, polycyclic aromatic hydrocarbons; PCBs, polychlorinated biphenyls; BKMEs, bleached kraft pulp and paper mill effluents; B(a)P, benzo(a)pyrene; DEHP,diethylhexyl phthalate. References: (1) Arnold et al., 1995; (2) Au et al., 1999; (3) Braunbeck and Volkl, 1991; (4) Bucher et al., 1992; (5) Cajaraville et al., 1990a; (6)Cajaraville, 1991; (7) Cajaraville et al., 1997; (8) Cancio et al., 1998; (9) Cancio et al., 1999; (10) Donohue et al., 1993; (11) Ibabe et al., 2001; (12) Ibabe et al.,submitted-a; (13) Krishnakumar et al., 1997; (14) Mather-Mihaich and Di Giulio, 1991; (15) Orbea et al., 1999a; (16) Orbea et al., 1999b; (17) Orbea et al., 2002a; (18)Orbea et al., 2002b; (19) Ortiz-Zarragoitia and Cajaraville, 2000; (20) Oulmi and Braunbeck, 1996; (21) Oulmi et al., 1995a; (22) Oulmi et al., 1995b; (23) Pedrajaset al., 1996; (24) Pretti et al., 1999; (25) Rocha et al., 1999; (26) Scarano et al., 1994; (27) Yang et al., 1990.


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1993; Ruyter et al., 1997) whereas no signs of peroxi-some proliferation are detected in sea bass Dicentrar-chus labrax injected with clofibrate (Pretti et al., 1999).It seems important to determine the risks to aquaticorganisms of environmental exposure to peroxisomeproliferators. Several reports have indicated increasedprevalence of liver tumours in fish inhabiting pollutedwaters although etiological agents have not been iden-tified in most cases (ICES, 1997).

PEROXISOME PROLIFERATION AS ANOVEL CELLULAR MARKER OF

ORGANIC POLLUTIONDuring the last decade, a considerable research ef-

fort has been devoted to developing sensitive earlywarning biomarkers of pollutant effects (McCarthy andShugart, 1990). Research has concentrated at the lowerlevels of biological organization, which might be ex-ploitable surrogates for changes at higher levels. Thus,changes at biochemical/molecular and cellular levelsare hypothesized to occur early and to anticipatechanges at higher levels of the biological hierarchy. Forinstance, in the laboratory experiment where musselswere exposed to the WAF of different crude and lubri-cant oils, changes indicating cellular damage at day21 of exposure were significantly correlated with lowvalues of growth and condition indices at day 91 ofexposure (Cajaraville et al., 1993).

Our research group has been involved in the devel-opment of pollution biomarkers at the cellular level(Cajaraville et al., 1995, 1998, 2000). The use of cellularapproaches to measure exposure to pollutants and pol-lutant effects has several advantages. First, cellularapproaches allow detection of cell-specific changes orresponses. This is crucial in several organs and tissueswith heterogeneous cell composition where only a cer-tain cell type may be responsive to pollutants (e.g., the

digestive gland of bivalve molluscs). It is also impor-tant in cases where pollutants affect cell type compo-sition (e.g., replacement of digestive cells by basophiliccells in stressed molluscan digestive glands, Cajara-ville et al., 1990b). Second, through the use of a set ofcellular markers a global view of pollutant effects canbe achieved as biochemical/molecular processes all be-come integrated at the cellular level. Thus, cells pro-vide a link between biochemical/molecular events andphysiological or whole-animal events occurring withinliving organisms.

As reviewed above, peroxisome proliferation hasemerged as a possible biomarker of exposure to severalorganic pollutants including phthalate ester plasticiz-ers, PAHs and oil derivatives, PCBs, certain pesticides,bleached kraft pulp and paper mill effluents, alkylphe-nols, and estrogens. Although this proposition has beeninvestigated in few aquatic species, mainly the marinemussel and a few species of fish, this novel cellularbiomarker appears to be linked significantly to pollut-ant exposure. In fish, as in other vertebrates, cyto-chrome P450-based biomarkers are generally employedas exposure biomarkers for organic pollutants. Expo-sure to organic contaminants including PAHs andPCBs induces the activity of enzymes of the cytochromeP450 system in fish, particularly of the CYP1A subfam-ily, involved in biotransformation of endogenous com-pounds and xenobiotics (Goksøyr and Forlin, 1992).Exposure to typical peroxisome proliferators such ashypolipidemic drugs induces the activity of the CYP4Asubfamily in rodents, involved in the �-oxidation offatty acids (Simpson, 1997). CYP2 proteins identifiedin fish (CYP2M1 and CYP2K1) are similarly inducedon exposure to hypolipidemic drugs (Haasch, 1996; Ha-asch et al., 1998). The induction of CYP1A and CYP4Aoccurs through the cytosolic Ah (arylhydrocarbon) re-ceptor and the nuclear receptor PPAR, respectively.

Fig. 2. Light micrographs of PPAR� immunostained sections ofparaformaldehyde-fixed paraffin-embedded tissues of zebrafish. Anti-gen retrieval was performed combining microwaving and trypsin di-gestion. Tissue sections were incubated with a rabbit anti-mousePPAR� antibody followed by a peroxidase coupled anti-rabbit second-ary antibody. Positive labeling was developed using the NovaRed kit

from Vector (Burlingame, CA). A: Expression of PPAR� is shown inenterocytes, pancreatic cells, proximal tubules of kidney, and ovo-cytes. B: In the liver, PPAR� immunolabeling is found in manyhepatocytes, both in the nuclei (arrows) and cytoplasm. Scale bars �100 �m. [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]



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The presence of multiple forms of CYPs has also beenindicated in mussels, including a putative CYP1A thatcould be contaminant-inducible (Wootton et al., 1995;for a review, see Livingstone et al., 2000). However, theresponse of the cytochrome P450 system is not alwaysconsistent in molluscs (Cajaraville et al., 2000; Canovaet al., 1998; Livingstone et al., 2000; Porte et al., 2001;and references cited therein). Thus, peroxisome prolif-eration acquires particular relevance as a pollutionbiomarker in mussels and other molluscs, as a possiblealternative to cytochrome P450-based biomarkers.

One crucial issue when applying the biomarker ap-proach in environmental pollution assessment is to beable to distinguish pollution-related changes in bi-omarkers from “natural” variations (Cajaraville et al.,2000). Indeed, several biotic (e.g., developmental stage/age, gender, nutritional status, reproductive cycle) andabiotic factors (e.g., water temperature, salinity andpH, dissolved oxygen, season) can fundamentally influ-ence peroxisomal responses (Table 1). It has recentlybeen reported that peroxisomal enzyme activities andperoxisomal structure vary depending on tidal leveland season (Cancio et al., 1999; Ibabe et al., submit-ted-a; Orbea et al., 1999b, 2002b). Thus, catalase andAOX activities and Vvp experience pronounced seasonalfluctuations, showing maximum values at spring-sum-mer months and minimum values at autumn-wintermonths. Vvp was positively correlated with AOX andcatalase activities. On the other hand, these three pa-rameters were negatively correlated with the volumedensity of neutral lipids in digestive gland tubules.These seasonal changes have been suggested to berelated to the reproductive cycle (Cancio et al., 1999).Season-dependent changes have also been reported infish. In gray mullet Mugil cephalus, peroxisomal Vvpand size increase in summer in comparison with winter(Orbea et al., 1999a, 2002b). In the brown trout (Salmotrutta), seasonal differences have been found in perox-isomal volume and surface densities and size (Rocha etal., 1999). Male trout showed higher peroxisomal den-sities in February and September in comparison withMay when both genders presented similar values.Meanwhile females presented the lowest values in Sep-tember (Rocha et al., 1999). The detailed knowledge ofseasonal variations in peroxisomal parameters is es-sential to be able to detect changes induced by contam-inants. In addition, peroxisomes may respond differ-ently to pollutants depending on the time of the year.

Orbea et al. (1999b) found that seasonal variations inperoxisomes differed in two different populations ofmussels. Population-specific responses can be ascribedto several factors including (1) genotypic differencesbetween populations, (2) differences in the reproduc-tive cycle, nutritional status or other factors specific ofeach population, and (3) environmental variables char-acterizing the area inhabited by each population in-cluding temperature, presence of contaminants, etc. Inthe study of Orbea et al. (1999b), mussels inhabiting arelatively clean reference area showed more markedseasonal variations than those living in a chronicallycontaminated station. Ideally, biomonitoring programsinvolving different populations living in different sitesshould previously characterize each population withregard to population genetics, nutritional status, repro-ductive cycle and environmental conditions in order to

attain comparable biomarker responses. Transplantexperiments in which native organisms are comparedwith transplanted ones (see below) can also help avoidmisinterpretations due to comparisons of different pop-ulations of aquatic organisms.

In addition to seasonal fluctuations and population-specific responses, gender of animals could be anotherpossible confounding factor. In mussels, no differencesin AOX activity or Vvp were observed between malesand females sampled at different seasons (Ibabe et al.,submitted-a). Similarly, no significant gender-relateddifferences were detected in zebrafish with respect toAOX activity and levels of liver PPAR� protein expres-sion (Ibabe et al., 2001). These results indicate thatPPAR� and its target gene AOX are not regulateddifferentially in sexually mature adult male and femalezebrafish, which is in agreement with data obtained inmammals (Beier et al., 1997; Braissant et al., 1996;Lemberger et al., 1996a). However, significant differ-ences between genders have been reported in browntrout (Rocha et al., 1999). It remains to be establishedif environmental pollutants can induce peroxisome pro-liferation differentially in male and female aquatic or-ganisms, as reported in rats treated with the hypolipi-demic drug bezafibrate (Fahimi et al., 1982). In mousetreated with trichloroethylene, induction of certain en-zymes and PPAR� protein and mRNA was higher inmale hepatocytes than in female ones (Nakajima et al.,2000).

Apart from gender-related differences, significantvariability in levels of PPAR� has been detected amongdifferent zebrafish individuals and also among hepato-cytes within a given individual (Fig. 2, Ibabe et al.,2001). As reported before for mouse and rat liver, in-terindividual variability could be related to differencesin hormonal status since PPAR� expression is tightlyregulated by various hormones like insulin and glu-cocorticoid hormones (Lemberger et al., 1994, 1996a,b;Shalev et al., 1996). The reproductive state could alsoinfluence PPAR expression as signaling cross-talk be-tween PPAR and estrogen receptor has been reported(Lemberger et al., 1996a). Finally, preliminary dataindicate significant differences in AOX activity andPPAR� protein levels in zebrafish depending on devel-opmental stage (Ibabe et al., 2001). Differences in liverperoxisomal size and Vvp have also been observed inmullets of different ages, adult mullets displayinghigher values than juveniles (Orbea et al., 1999a).

APPLICATION OF PEROXISOMEPROLIFERATION AS BIOMARKER IN

A TRANSPLANT EXPERIMENTFollowing this overview of evidence indicating the

possible use of peroxisome proliferation as pollutionbiomarker, a recent example of its application in atransplant experiment using mussels as sentinel or-ganisms is presented. The transplant experiment wascarried out in autumn (November to December) whenbasal levels of peroxisomal �-oxidation enzyme activi-ties and Vvp are low (Cancio et al., 1999; Orbea et al.,1999b). The experiment was run in two nearby estuar-ies of the Basque coast in the Bay of Biscay (Fig. 3).Txatxarramendi is located in the Biosphere’s Reserveof Urdaibai and it was selected as the reference stationbecause previous studies demonstrated low levels of


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pollution by organic xenobiotics such as PAHs andPCBs (Orbea et al., 2002b). The station designated asArriluze is a small leisure port in the mouth of NerbioiRiver, heavily polluted over several decades. Althoughcontaminant levels have been reduced in recent yearsmainly due to the industrial crisis and implementationof new policies, high levels of persistent pollutants arestill found in the area (Pagola-Carte and Saiz-Salinas,2001; Saiz-Salinas, 1997). In the experiment, musselsfrom Txatxarramendi were transplanted in cages toArriluze and sampled after 2 and 21 days. Simulta-neously, mussels from Arriluze were transplanted toTxatxarramendi for 2 and 29 days. Mussels from Arri-luze and Txatxarramendi were also caged in their re-spective stations as handling controls.

Chemical analyses of body burdens of PAHs andPCBs were performed in mussel soft tissues. As shownin Figure 3, mussels originally from Arriluze showedabout 40-fold higher concentrations of PAHs and 3-foldhigher concentrations of PCBs than animals from Tx-atxarramendi. Mussels transplanted to Arriluze for3 weeks drastically increased their PAH and PCB bodyburdens reaching values similar to those of native an-imals. Conversely, animals transplanted from Arriluzeto Txatxarramendi for 4 weeks showed reduced levelsof PAHs by one order of magnitude, but PCBs were notdecreased (Fig. 3).

Peroxisome proliferation was assessed by measuring(1) AOX activity in mussel digestive gland homoge-nates and (2) Vvp in diaminobenzidine (DAB)-stainedcryostat sections as described in Cancio et al. (1998)and Orbea et al. (1999b). Mussels originating fromArriluze, the polluted area, showed 2–3-fold higherAOX activity than animals from Txatxarramendi inboth samplings (Fig. 4A). When animals were trans-planted from Txatxarramendi to Arriluze, with in-creased body burdens of organic xenobiotics, a concom-itant significant increase in peroxisomal AOX activitywas recorded and by day 21 both native and trans-planted animals showed the same activity levels. Con-versely, in animals transplanted from Arriluze to Tx-

atxarramendi for 29 days, AOX activity decreaseddrastically reaching levels of native animals from Tx-atxarramendi (Fig. 4B). At the beginning of the exper-iment, mussels from Arriluze showed 4-fold higher Vvpthan animals from Txatxarramendi (Fig. 4B). After2 and 29 days in Txatxarramendi, mussels originatingfrom Arriluze recorded a significant decrease in Vvp.However, in animals from Txatxarramendi, no changesin Vvp were detected when transplanted to Arriluze(Fig. 4B).

In conclusion, peroxisome proliferation measured asincreased Vvp and AOX activity appears to be a sensi-tive biomarker to assess exposure of mussels to organicpollution. When comparing changes in AOX and Vvp, itappears that increases in AOX activity preceed in-creases of Vvp in response to changes in PAH and PCBbody burdens. Most importantly, the transplant exper-iment has demonstrated for the first time the revers-ibility of the peroxisome proliferation response in mus-sels, which is a main feature of a reliable biomarker(Table 1).

MEASUREMENT OF PEROXISOMEPROLIFERATION IN AQUATIC ORGANISMSThe implementation of the biomarker approach for

biomonitoring and regulatory purposes greatly de-pends on the establishment of well-defined protocolstogether with intercalibration and quality assuranceprogrammes (Cajaraville et al., 2000). Up to now, dif-ferent laboratories have used different methods tomeasure peroxisome proliferation in aquatic organ-isms. Generally, activity of peroxisomal �-oxidation en-zymes (AOX, PH, and thiolase) or other enzymes in-ducible by peroxisome proliferators is measured as in-dicative of peroxisome proliferation (Cancio et al.,1998; Haasch et al., 1998; Mather-Mihaich and Di Giu-lio, 1991; Pedrajas et al., 1996; Scarano et al., 1994).Alternatively, peroxisomal protein levels have beenmeasured by immunoblotting or immunohistochemis-try coupled to image analysis (Cajaraville et al., 1998,

Fig. 3. Map of the Biscay coast representing the estuaries wherethe transplant experiment was carried out. PAH and PCB concentra-tions in native (italics) and transplanted mussels are expressed inng/g wet weight (n � 1).

Fig. 4. A: Peroxisomal acyl-CoA oxidase activity (n � 6) and (B)peroxisomal volume density (n � 10) of native and transplantedmussels. Vertical segments represent standard errors. *Significantdifferences between transplanted groups at both samplings and theirrespective native group at the first sampling; #Significant differencesbetween transplanted groups and their respective destination groupat each sampling period; $Significant differences between the twosampling periods for the same group according to the Student’s t-test.Statistical significance was established at P � 0.05.


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Orbea et al., 2002a,b). For instance, Ackers et al. (2000)have proposed that levels of PMP70, a peroxisomalintegral membrane protein, is a suitable indicator ofperoxisome proliferation in fish. More recently, molec-ular tools have been developed to measure induction ofexpression of specific genes by using RNA or DNAtechnology. But, as increases in peroxisomal volumeand induction of peroxisomal enzymes do not alwaysoccur in parallel (Baumgart et al., 1990; Lazarow et al.,1982), occurrence of peroxisome proliferation can onlybe confirmed by morphometric evaluation using eitherlight or electron microscopy. In aquatic organisms,some studies have used conventional electron micros-copy (Bucher et al., 1992; Au et al., 1999) or electronmicroscopy of diaminobenzidine (DAB)-stained mate-rial (Arnold et al., 1995; Braunbeck and Volkl, 1991;Yang et al., 1990) not always combined with morpho-metric measurements. We have used DAB histochem-istry on cryostat sections in combination with a stereo-logical technique to measure peroxisomal volume, sur-face and numerical densities (Cajaraville et al., 1997;Cancio et al., 1998; Fahimi and Cajaraville; 1995).Krishnakumar et al. (1995, 1997) have also used DAB-stained cryostat sections but have assessed peroxisomeproliferation semi-quantitatively. Thus, there is a needto define a simple and reliable standardized protocol tomeasure peroxisome proliferation in aquatic organismsif this biomarker is to be used widely and to become thesubject of intercalibration exercises. Such protocolshould combine morphological and biochemical/molec-ular endpoints since peroxisome proliferation mightoccur without changes in biochemical parameters andvice versa.

As a rapid and specific marker of enzyme inductionby peroxisome proliferators, we propose the measure-ment of AOX, the first and rate-limiting enzyme of theperoxisomal �-oxidation system, whose transcription isregulated by PPAR�. The second enzyme of the perox-isomal �-oxidation system PH is also considered a sen-sitive marker of peroxisome proliferation (Reddy andLalwani, 1983). The expression of the most abundantperoxisomal enzyme catalase is not regulated byPPARs and, indeed, catalase activity is only slightlyinduced or even inhibited by peroxisome proliferators(Cancio et al., 1998; Fahimi and Cajaraville, 1995;Reddy and Mannaerts, 1994). Hence, catalase mea-surements cannot be considered good markers of per-oxisome proliferation. As to AOX, fatty acyl-CoA oxi-dase activity is easily measured in aquatic organismsusing palmitoyl-CoA as substrate according to themethod developed by Small et al. (1985) where palmi-toyl-CoA oxidation is followed by measuring peroxiso-mal H2O2 production. Cyanide-insensitive AOX can bemeasured following production of NADH after inhibi-tion of mitochondrial activity with cyanide to preventNADH reoxidation (Lazarow and De Duve, 1976). AOXactivity can be measured either in homogenates of fishliver or molluscan digestive gland or in enriched per-oxisomal fractions. In mussels, AOX activity per mg ofprotein is similar or lower using peroxisomal fractions(unpublished results) and, thus, the use of homoge-nates is recommended, since cell fractionation is time-consuming and requires specialized equipment andtraining. When homogenates are used, activity mea-surements need not be performed on freshly dissected

tissues but can be equally well performed on frozentissues (Cancio et al., 1998; Orbea et al., 2002a), whichis very convenient for biomonitoring studies in thefield.

Immunochemical techniques including immunoblot-ting and immunohistochemistry are specific and sensi-tive methods to measure quantitative alterations inlevels of peroxisomal proteins (Orbea et al., 2002a,b).Immunohistochemistry could be more sensitive be-cause labeling is measured only in positive cells, not inthe whole organ (Orbea et al., 2002b). The main prob-lem of these immunotechniques is the lack of specificantibodies against peroxisomal proteins of aquatic spe-cies. Nevertheless, peroxisomal proteins appear to behighly conserved in evolution and, thus, antibodiesagainst mammalian catalase, AOX, PH, and PMP70cross-react with the corresponding proteins in liver ofdifferent fish species (Ackers et al., 2000; Orbea et al.,1999a) or mussel digestive gland (Cajaraville et al.,1992; Cancio et al., 2000a). Commercial antibodies alsoexist against mammalian PPARs, which cross-reactwith the corresponding zebrafish receptors (Fig. 2; Ib-abe et al., 2001, submitted-b). Another promising ap-proach for application of immunochemical techniquesin aquatic organisms is the assessment of peroxisomalprotein levels using antibodies against the tripeptideserine-lysine-leucine or SKL (Fig. 5). The SKL aminoacid sequence or its variants appears in the carboxy-terminus of several peroxisomal matrix proteins anddirects them for import into peroxisomes (Gould et al.,1988). This peroxisomal targeting sequence is highlyconserved in evolution (Keller et al., 1991). Levels ofspecific peroxisomal proteins, PPAR, or SKL-contain-ing proteins, can be determined quantitatively by den-sitometry (Orbea et al., 2002b).

Similarly, RNA or cDNA probes available for mam-malian organisms (Fahimi and Baumgart, 1999) couldbe used in aquatic organisms for in situ hybridizationand Northern blotting studies. In certain cases, itmight be necessary to construct species-specific probessince some mammalian probes have been shown not tocross-hybridize with molluscan mRNA molecules (un-published results). An advantage of mRNA detection ofperoxisomal proteins is the rapid induction of tran-scriptional systems in comparison to the lag time nec-essary for changes to occur in post-transcriptionalevents. Assays for transactivation of PPAR� receptorare also available (Maloney and Waxman, 1999) andcould be used to show the involvement of PPARs inperoxisome proliferation in aquatic species. DNA mi-croarray technology could provide a powerful approachto measure effects of peroxisome proliferators inaquatic organisms. This technique could be used tosimultaneously monitor the expression of the battery ofgenes related to peroxisome proliferation (PPARs andother nuclear receptors, peroxisomal, microsomal, andmitochondrial enzymes) and regulation of cell growth(protooncogenes, tumor-suppressor genes).

For the microscopic evaluation of peroxisome prolif-eration, measurement of volume, size, and number ofperoxisomes should be performed using quantitativemicroscopy or computer-assisted image analysis. Forthis, peroxisomes are specifically stained by using en-zyme histochemical or immunohistochemical methodsfor detection of marker enzymes. The most widely used


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is the alkaline DAB method for catalase demonstration(Deiman et al., 1991; Fahimi, 1969) applicable in resin-embedded material at both light or electron micro-scopic levels. This method works well in fish liver, withdistinct staining of peroxisomes in hepatocytes (Fig. 5;Orbea et al., 1999a), but gives only weak staining ofperoxisomes in mussel digestive gland. The alkalineDAB technique has been recently modified for applica-tion in cryostat sections of frozen mussel digestivegland (see Fig. 1; Cajaraville et al., 1993, 1997; Orbeaet al., 1999b). Due to the small size of peroxisomes inmussels and due to DAB staining of structures otherthan peroxisomes (i.e., lipofuscins), direct measure-ment of peroxisomes by image analysis is not possibleand a manual point counting stereological techniquehas been used instead (Cajaraville et al., 1997; Fahimiand Cajaraville, 1995). When compared to electron mi-croscopy, morphometry at the light microscopic levelseems to underestimate proliferation (Beier and Fa-himi, 1987), thus improvement of the morphometricassessment of peroxisome proliferation is still needed.

CONCLUDING REMARKS ANDFUTURE PROSPECTS

Recent studies have advanced our knowledge greatlyon the occurrence of peroxisome proliferation inaquatic organisms exposed to certain environmentalpollutants but many questions still remain due to thecomplexity of the systems concerned. Indeed, morethan three decades of research in classical toxicologyhave focused mainly on studying drugs of therapeuticuse and their peroxisome-proliferating effects in a fewspecies, with emphasis in humans, whereas environ-mental toxicologists face a problem of huge propor-tions, with an almost infinite number of compounds

and species involved. This makes the individual eval-uation of each compound in each species impossible,but detailed toxicological investigations should be un-dertaken in sentinel species at least with priority con-taminants with suspected peroxisome proliferationability. Most importantly, aquatic organisms are ex-posed simultaneously to complex mixtures of contami-nants that cannot be reproduced in the laboratory. Thishighlights the importance of field studies, includingtransplant experiments, in combination with the bi-omarker approach to establish exposure and effects ofenvironmental pollutants on the biota.

The magnitude of peroxisome proliferation inaquatic organisms may not vary only with differentconcentrations and times of exposure for each com-pound or mixture of compounds but also with severalbiotic and abiotic factors. In spite of the number ofvariables involved, both laboratory and field studieshave indicated the occurrence of peroxisome prolifera-tion in different fish or bivalve mollusc species treatedwith phthalate ester plasticizers, PAHs and oil deriv-atives, PCBs, certain pesticides, bleached kraft pulpand paper mill effluents, alkylphenols, and estrogens.Hence, peroxisome proliferation could be used as anexposure biomarker for the latter pollutants. Furtherresearch on the potential of peroxisome proliferation asan exposure biomarker should be concentrated in mus-sels because the generally employed cytochrome P450-based exposure biomarkers, for organic pollutants infish, give unreliable results in mussels (Cajaraville etal., 2000; Canova et al., 1998; Porte et al., 2001).

Peroxisome proliferation involves strong and specificactivation of gene expression by a mechanism involvingactivation of PPARs. For the majority if not all perox-isome-proliferating environmental pollutants, it is not

Fig. 5. Electron micrographs of mullet (Mugil cephalus) liver ul-trathin sections. A: Glutaraldehyde fixed, osmium post-fixed epoxyresin-embedded liver showing peroxisomes positively stained with theDAB technique. Scale bar � 1 �m. B: Glutaraldehyde plus formalde-hyde fixed, Lowicryl-embedded liver incubated with the anti-SKL

antibody and the protein A gold technique. Colloidal gold particles(10 nm) show the presence of the SKL sequence in peroxisomal matrixproteins. P, peroxisome; ER, endoplasmic reticulum; M, mitochondria.Scale bar �1.5 �m.


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known whether they bind to PPARs and/or act as tran-scriptional activators. Also it is not known whetherPPAR subtypes are inducible in aquatic organisms ex-posed to peroxisome proliferating pollutants in theirenvironment. A recent report indicates that expressionof the PPAR� subtype is increased in salmon hepato-cytes treated with peroxisome proliferators (Ruyter etal., 1997). Target genes containing PPREs are notknown in fish except for genes of glutathione-S-trans-ferase in the plaice (Leaver et al., 1997). These areup-regulated after administration of peroxisome prolif-erators, and their products appear to be efficient in theconjugation of some of the end-products of lipid peroxi-dation (Leaver et al., 1997; Leaver and George, 1998).A question that needs to be answered is whether per-oxisome proliferation in mussels and other inverte-brates is mediated by PPARs or similar ligand-acti-vated transcription factors.

Apart from peroxisome proliferation, the pleiotropiceffects induced by peroxisome proliferators in rodentsinclude increased DNA synthesis and cell division(Roberts et al., 1995). This feature could contribute tothe ability of peroxisome proliferators to induce livertumours in rodents (James and Roberts, 1995). Otherfactors such as increased oxidative stress and DNAdamage (Reddy and Lalwani, 1983; Yeldandi et al.,2000), promotion of initiated liver foci (James and Rob-erts, 1995), and suppression of apoptosis (Roberts etal., 1995) could also contribute. To the best of ourknowledge, there are no studies addressing the possi-ble hepatocarcinogenic effect of peroxisome prolifera-tors in aquatic organisms and thus this is an importantgap to be filled in future studies. The potential delete-rious effects on reproduction, development, and growthare also unknown (Table 1), providing a challenge forfuture investigations. In rodents peroxisome prolifera-tors are known to adversely affect reproduction anddevelopment. For instance, phthalate esters are estro-genic (Jobling et al., 1995) and produce adverse repro-ductive effects disrupting normal male development(Wine et al., 1997). These data open a promising newfield of research aiming to establish possible connec-tions between peroxisome proliferation and endocrinedisruption, perhaps based on cross-talk betweenPPARs and estrogen receptors (Ortiz-Zarragoitia andCajaraville, 2000).

ACKNOWLEDGMENTSThanks are due to Prof. S. Subramani (University of

California, San Diego) for the antibody against SKL.

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AQ: 3

AQ: 3


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AQ1: Provide year. See query #2 on disk

AQ2: “ CICYT, Spanish Ministry of Science and Technology” Correct sponsor name?

AQ3: If in press or published, provide year here and update in References with volume # and pages. Pleasebe sure that Ibabe et al. “submitted a and b are both cited in text with years, and that full references areprovided in Reference section if they are in press or have been published. If both submitted a,b are 2001,they should be cited as 2001b and c (there is already a 2001 citation, which should then become 2001a).

AQ4: 1.5 correct (not 1,5)?



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Microscopy Research and Technique - UPV/EHU › europeanclass2003 › CAJperprolif-MRTproof.pdf · that ﬁbrates, used as hypolipidemic drugs to lower the lipid levels in serum of