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Environmental Toxicology and Chemistry, Vol. 21, No. 11, pp. 2434–2444, 2002q 2002 SETAC

Printed in the USA0730-7268/02 $9.00 1 .00

FUNCTIONAL AND MORPHOLOGICAL CHANGES OF LYSOSOMES AS PROGNOSTICBIOMARKERS OF TOXIC LIVER INJURY IN A MARINE FLATFISH

(PLATICHTHYS FLESUS (L.))

ANGELA KOHLER,*† ELLEN WAHL,† and KARIN SOFFKER‡†Alfred Wegener Institute for Marine and Polar Research, Department of Ecophysiology and Ecotoxicology, P.O. Box 120161,

27515 Bremerhaven, Germany‡Heinrich-Lehmann-Straße 2, 31542 Bad Nenndorf, Germany

(Received 1 February 2002; Accepted 13 May 2002)

Abstract—Lysosomes are cell organelles in which macromolecules are recycled and that protect cells against toxins. In the presentstudy, we apply histopathological and histochemical techniques in parallel with analytical chemistry of organochlorines and heavymetals in livers, with and without histopathological lesions, of the flatfish Plathichthys flesus (L.). The fish were caught along apollution gradient on the German North Sea coast. We analyze critically whether changes in morphology and function of lysosomes,reflecting the onset and progression of toxipathic lesions in the liver of flounder, are appropriate for the rapid application inbiological-effect monitoring programs such as BEEP (European Union Project in the 5th Framework Biological Effects of Envi-ronmental Pollutants in Marine Coastal Ecosystems). Livers collected from wild flounder were graded into four categories of lesions.Lysosomal stability was plotted against progressive lesions and contaminant levels in the same livers. Lysosomal membrane stabilitywas already decreased at the onset of liver anomalies that are regarded as reversible. Reduced membrane stability also persistedin degenerative liver lesions and during carcinogenesis in liver parenchyma surrounding foci and tumors. Decreased lysosomalmembrane stability coincided with increased size of lysosomes and increased lipid content during the progression toward degen-eration. As predicted by the resistant-cell hypothesis, in proliferating cancer cells of adenomas and carcinomas, the highest valuesof membrane stability were detected. Concentrations of organochlorines such as hexachlorcyclohexane isomers, DDT metabolites,and polychlorinated biphenyls were correlated with reduced lysosomal membrane stability in noncancerous hepatocytes. On thebasis of these findings, we recommend applying the lysosomal membrane stability test as an expertise-independent and rapidprognostic biomarker for toxically induced liver injury and carcinogenesis in monitoring programs.

Keywords—Lysosomes Prognostic biomarker Liver injury Carcinogenesis Flatfish

INTRODUCTION

The evolution of life in extreme biotopes such as hydro-thermal environments, characterized by high levels of metalsand hydrogen sulfide, was possible because of the capacity oflysosomes to trap foreign toxic compounds and bind them ina nontoxic form [1,2]. Lysosomes are multifunctional cell or-ganelles involved in macromolecular catabolism as well as inprotection against toxic compounds and viral and bacterialinfection [3,4]. Another aspect is intralysosomal lysis of en-dosymbiotic chemoautotrophic bacteria, making these micro-organisms available as nutrients for their bivalve hosts livingin biotopes reduced in food availability [5].

The lysosome, with its acidic interior, is in the center ofthe endosomal-lysosomal system, which is continuous withGolgi-derived vesicles with newly synthesized enzymes, theprimary lysosomes. Autophagic and heterophagic vacuolesfuse with primary lysosomes, forming secondary lysosomesand residual bodies that accumulate indigestible material.

In human diseases, lysosomal storage disorders have seri-ous consequences and show the relevance of lysosomal func-tion for cellular homeostasis. These disorders provoke intra-lysosomal accumulation of degradative resistant b-amyloid insecondary lysosomes, leading to senile plaques in Alzheimerdisease [6] or to other storage abnormalities that cause enzymedeficiencies of alpha-galactosidase in Fabry’s disease and acid

* To whom correspondence may be addressed(akoehler@meeresforschung.de).

beta-glucosidase in Gaucher’s disease [7]. During invasivegrowth and metastasis of cancer, release of lysosomal enzymes,such as extracellular proteinases and/or cathepsin B, plays animportant role [8,9]. Apoptosis, a major type of active celldeath, which plays an important role in the elimination ofsurplus cells during embryogenesis, metamorphosis, and main-tenance of tissues, is initiated and regulated by a whole familyof lysosomal enzymes [10].

Techniques to determine lysosomal dysfunction have beendeveloped primarily in human medicine [11] and have beentransferred to environmental research. They were first appliedin blue mussel (Mytilus edulis) [12–14] and later in variousmarine fish species such as flounder, dab, turbot, and rabbitfish[15–19]; to freshwater insects such as the caddisfly larvae [20];and to terrestrial species such as earthworms [21] for moni-toring effects of anthropogenic toxins.

In general, morphological changes and membrane stabilityof lysosomes, determined by reduced latency of lysosomalenzymes as an index of lysosomal function, are monitored inmussel-watch programs in Europe and in the United States asbiomarkers of the effects of pollution. Usually, hepatopancreasand blood cells of marine invertebrates, such as the blue mus-sel, the green mussel, and winkles, are used (for review, seeMoore et al. [22]).

Less is known about lysosomal perturbations in the liverof fish and their potential to reflect hepatic pathologies, whichare much more complex than those observed in invertebratesand closely resemble toxipathic lesions and carcinogenesis of

Lysosomes as prognostic biomarkers in fish Environ. Toxicol. Chem. 21, 2002 2435

the mammalian liver [23–26]. Histochemical assays to detectlysosomal changes could serve as rapid and cost-effective bio-markers for pinpointing effects of toxic exposures that presentrisks for the health and survival of fish species. Though his-topathological diagnosis is regarded as one of the most reliablemethods for detecting cell and tissue lesions related to toxicand carcinogenic exposure [27–32], this approach is generallytoo time consuming, too expertise dependent, and too costlyfor an initial screening of the health of aquatic organisms atrisk in areas of suspected contaminant input.

Lysosomal perturbations as biomarkers will be of particularuse if they can be shown to be precursors of pathology becausethis will relate directly to the risk potential of contaminantexposure. Furthermore, it is essential to analyze whether clearlinks exist between cellular responses, contaminant exposure,and contaminant accumulation in liver. Therefore, we choseflounder (Platichthys flesus (L.)), the candidate of choice formonitoring coastal and estuarine environments [17,33,34](BEEP—Biological Effects of Environmental Pollution in Ma-rine Coastal Ecosystems, European Union 5th Framework).We diagnosed toxipathic liver lesions, for which lysosomaldisorders in hepatocytes were determined, and related theseparameters to the liver concentrations of contaminants (po-lychlorinated biphenyl [PCB] congeners, octachlorostyrene,hexachlorobenzene, DDT metabolites, hexachlorcyclohexaneisomers, and heavy metals Hg, Cd, and Pb) that characterizethe pollution situation along the chosen transect of the NorthSea coast.

Cytochemical and immunohistochemical methods, advan-tageous as biochemical approaches because tissue homoge-nization can be avoided, are used to relate specific metabolicchanges in lysosomes of hepatocytes to the various types ofliver lesions and stages of carcinogenesis. Furthermore, ourfindings are compared with the lysosomal perturbations re-ported in invertebrates.

We applied this multidisciplinary approach in order to de-termine whether alterations of lysosomal properties reflect tox-ipathic lesions in livers of fish living in environments subjectto anthropogenic activities and to determine whether the ap-proach can be used as a rapid and objective diagnostic andprognostic tool in risk assessment of effects of environmentalpollution.

MATERIALS AND METHODS

Flounder (P. flesus) were caught from May to Septemberalong a contaminant gradient of the German North Sea coastfrom the Elbe estuary, the most polluted river in Europe [35],northward to the Eider estuary.

Only female individuals were evaluated for this study be-cause they display significantly higher frequencies of toxi-pathic lesions, including liver cancer, than males (Kohler etal., unpublished data). After 20-min hauls with a dragnet (70-mm mesh width), fish were processed according to the guide-lines developed during the BEQUALM workshop(www.cefas.co.uk/bequalm/default.asp) [22,32,36]. Flounderbetween 18 and 25 cm, representing female juvenile prespawn-ing, and female adults between 25 and 48 cm were selected.During visual inspection, juvenile females rarely showed in-dications of neoplastic changes in livers, while in older in-dividuals, neoplastic changes of varying size were frequentlyidentified macroscopically.

Enzyme histochemistry

Lysosomal latency. The functional integrity of the limitingmembranes of lysosomes that guarantees the enclosure of deg-radative lysosomal enzymes inside the organelle (latency) wasdetermined histochemically by the lysosomal latency test. Liv-er pieces were dissected and quick frozen in 2708C super-cooled n-hexane. Additionally, in the case of macroscopic di-agnosis of neoplastic changes, the lesion and the surroundingextralesional parenchyma were dissected. Serial cryostat sec-tions (10 mm thick), cut at a cabin temperature of 2258C, wereexposed to artificial acid labilization at 0, 2, 4, 6, 8, 10, 15,20, 25, 30, 35, 40, 45, and 50 min in 0.1 M citrate buffer, pH4.5, in a shaking water bath at 378C. After labilization, allsections were incubated in medium containing 20 mg naphtholAsBiN-acetyl-b-D-glucosamide (Sigma, St. Louis, MO, USA)as substrate for the lysosomal marker enzyme N-acetyl-hex-osamidase with a pH optimum of 4.5. The substrate was dis-solved in methoxy ethanol, 3.5 g low-viscosity polypeptide(Polypep P5115, Sigma) dissolved in 50 ml 0.1 M citrate buffer(pH 4.5), with 2.5% NaCl added shortly before incubation at378C for 15 min. Visualization of the enzyme-substrate com-plex was achieved by a 9-min postcoupling reaction using 50mg of the diazonium salt Fast Violet B (Sigma) in 50 ml 0.1M phosphate buffer (pH 7.4). In order to avoid artifact bindingof Fast Violet B with Polypep, we thoroughly washed thesections in 3% NaCl. Afterward, rinsing in running tap waterfor 10 min was followed by 15 min of fixation in Baker’sformol [37]. After an additional three rinses in tap water, sec-tions were mounted in glycerine gelatin. Adequate tissue ma-terial for cutting the number of serial sections needed for thelabilization intervals was obtained only from neoplastic lesionslarger than 4 mm in diameter (hepatocellular adenoma andcarcinoma). For smaller foci, we obtained information only onthe extrafocal tissue.

The latency test for lysosomal enzymes determines the timeinterval of acid buffer exposure needed to destabilize the ly-sosomal membrane and thus uncover the latency of lysosomalenzymes. Penetration of the substrate through the lysosomalmembrane was assessed as the maximum staining intensity ofthe enzyme-substrate complex inside the lysosomes, as visu-alized by azocoupling with Fast Violet B and quantified bycomputer-assisted image analysis. The longer the acid bufferexposure needed to destabilize the lysosomal membrane, thehigher the membrane integrity of the lysosomes. In flounderliver, we generally identified two peaks of maximal stainingintensity, representing the membrane stability of secondarylysosomes (the first peak) and primary lysosomes (the secondpeak).

Storage disorders of lysosomes. Unsaturated neutral lipidswere detected by the Oil Red O technique according to BaylissHigh [38]. Cryosections were fixed in Baker’s formol, rinsedin distilled water, and placed in 60% triethyl phosphate for 1min, followed by staining in Oil Red O for 15 min at roomtemperature. The staining solution comprised 1 g Oil Red O,60 ml triethyl phosphate, and 40 ml distilled water. Distilledwater was added to the triethylphosphate before the dye wasadded. The mixture was heated to 1008C for 5 min underconstant stirring. After filtration, the stock solution was diluted3:2 before use. After staining, sections were washed in 60%triethyl phosphate for 30 s, washed again in distilled water,and mounted in aqueous Kaiser’s glycerol gelatin (9242;Merck, Darmstadt, Germany). Slides were stored in the dark.

2436 Environ. Toxicol. Chem. 21, 2002 A. Kohler et al.

Histology

For diagnosis of liver lesions, parallel to the analysis oflysosomal alterations, we dissected liver pieces directly ad-jacent to the area taken for the histochemical tests. Plasticembedding media instead of paraplasts were used in order toachieve optimal optical resolution for detailed pathologicaldiagnosis. Tissue pieces up to 10 3 5 mm were fixed in 4%Baker’s formol for a maximum of 24 h. Before embedding,the samples were rinsed two times in distilled water for 30min each time. Graded dehydration in acetone (40, 70, 100%)for 15 min each was followed by a 30-min rinse in monomersolution (80 ml 2-hydroxy-ethylmethacrylate, stabilized with200 ppm hydrochinone, 12 ml 2-butoxyethanol, and benzyl-peroxide with 25% H2O; Merck). After one change of themonomer solution, the monomer was allowed to penetrate intothe tissue at room temperature over night. Tissues wereblocked in a mixture of monomer solution and activator (10ml polyethylene glycol 200 and 1 ml N,N-dimethylaniline; 2ml activator was added to 92 ml of monomer) in plastic em-bedding molds. The blocks were polymerized at 48C (in orderto keep open the option of later use in immunohistochemistry)and left to rest for at least 1 d. Two-micrometer sections werecut using a rotation microtome and mounted on slides. Meth-acrylate sections (2 mm) were rinsed 3 3 5 min in aqua bidestand stained for 1 h in Gill’s hematoxylin (6 g hematoxylin/CI75290, 0.6 g sodium iodate, 52.8 g aluminium sulphate, 690ml distilled water, 250 ml ethylene glycol, and 60 ml glacialacetic acid, filtered before use; this solution is stable for oneyear). After rinsing for 20 min in running tap water, sectionswere stained in eosin for 1 min (156 ml of 96% alcohol, 20ml of 1% eosin, 2 ml of 1% phloxine, 1 ml of 100% aceticacid; this solution can be kept for only one week), dipped into80% isopropanol or alcohol for a few seconds, air dried, andmounted in Euparal.

Cryostat sections of 5-mm thickness, instead of 10 mm asfor enzyme studies, were cut for simultaneous histopatho-logical localization of the lesions seen in plastic sections.Cryostat sections were fixed for hematoxylin and eosin stain-ing in 5.4 ml 37% formaldehyde and 0.4 ml 50% glutaral-dehyde in 194.2 ml 0.1 M Sorensen’s phosphate buffer (pH7.2) [37] for 3 min, and sections were processed as describedabove.

Details of tissue preparations for the ultrastructural studiesof lysosomal changes at the electron-microscopic level aredescribed in detail elsewhere [28].

Criteria for histopathological diagnosis

Based on the similarities between the lesion types foundduring experimental chemical intoxication and carcinogenesisin mammals and fish, we used the pathology nomenclature ofmammalian histology as a standard for the classification oflesion types [23,25,27,31,39–43].

The diagnostic criteria are described in more detail in therecommendations of the International Council for the Explo-ration of the Sea Special Meeting on the use of liver pathologyof flatfish for monitoring biological effects of contaminants[44] and in the guidelines developed during the BEQUALMworkshop in Weymouth, including the standardized protocolsfor sample collection, tissue preparation, and quality assurance[32].

Chemical analysis in flounder liver

The selection of anthropogenic compounds analyzed inflounder liver was based on their ubiquitous presence and/oranalytical detection levels in various marine species, on evi-dence or the assumption of toxicity, and finally, on their es-sential roles in the contamination of the North Sea [45–47].Concentrations of organochlorines were measured and valuescalculated, on a wet weight basis, as described in detail inSoffker [48]. Lead and cadmium concentrations were analyzedin the same livers, and mercury was analyzed in muscle tissue,as described in Wahl et al. [17].

Statistics

For data evaluation, we applied the nonparametric Kruskal–Wallis test and a post hoc test of Nemenyii with a confidenceinterval of more than 95% (p , 0.05).

RESULTS

Histopathology

Briefly, we differentiated between healthy, normal liver;reversible early nonneoplastic toxicopathic lesions; irrevers-ible degenerative nonneoplastic lesions with severe storagedisorders (lipid) and extensive necrosis, foci of cellular alter-ation (clear, eosinophilic, and/or basophilic cells) of putativelypreneoplastic character; benign neoplasms (adenoma); and ma-lignant hepatocellular neoplasms (carcinoma).

The healthy, normal liver of flounder (P. flesus (L.)) con-sists of circular sinusoids surrounded by 6 to 10 hepatocytes(average diameter 15 mm) (Fig. 1A) with microvillous protru-sions into the space of Disse. Sinusoids are separated by stringsof two hepatocytes, in contrast with the mammalian liver,which is organized in lobuli or acini [49]. Under normal con-ditions, flounder liver stores more glycogen than lipids and ispoor in connective tissue, and the exocrine pancreas invadesalong the portal vein into the liver (hepatopancreas). At thereference site, we diagnosed healthy livers in 57% of the floun-der, while at the most contaminated site, only 0.1% of thelivers could be judged as healthy.

Early reversible nonneoplastic lesions comprised hepato-cellular and nuclear polymorphism and occasional fibrillar in-clusions (Fig. 1B) and were seen in 40.2% at the referencesite and in 29.2% close to the Elbe estuary. These changeswere associated with a moderate increase in lipid in the cy-toplasm, a decrease of glycogen, an increase of single-cellnecrosis and apoptosis, and increased numbers and sizes ofmacrophage aggregates.

Irreversible degenerative nonneoplastic lesions were char-acterized by excessive lipid accumulation in the cytoplasm(Fig. 1C) and lyosomes, as demonstrated by transmission elec-tron microscopy. Epithelial cells of bile ducts often showedhydropic vacuolization and proliferation of connective tissueof the surrounding externa. A frequent feature of this stage oftoxipathic lesion was cytoplasmic eosinophilic granules (Fig.1D), which ultrastructurally appeared to be phospholipidwhorls accumulating in lysosomes, indicative of toxically in-duced phospholipidosis (see Fig. 2). Commonly, fatty changewas associated with lytic necrosis (Fig. 1E). These lesion typeswere rare at the reference (3.8%) and occurred with 46.5%frequency at the most polluted site.

Foci of cellular alteration appear as discrete collections ofhepatocytes, the morphology and staining properties of whichdifferentiate them from the surrounding hepatic parenchyma.

Lysosomes as prognostic biomarkers in fish Environ. Toxicol. Chem. 21, 2002 2437

Fig. 1. Light microscopic photographs of hematoxylin and eosin-stained methacrylate sections of the liver of flounder with character-istic progressive lesion types. (A) Normal healthy liver consisting ofstrings of hepatocytes arranged around sinusoids (S), with lowamounts of lipid droplets in the cytoplasm. (B) Polymorphism ofhepatocytes and nuclei and loss of muralial structure of the liverparenchyma, diagnosed as reversible anomalies. (C) Dramatic accu-mulation of lipid droplets (LI, arrows) in cytoplasm with compressionof nuclei at the cell margin. (D) Accumulation of eosinophilic granulesrepresenting phospholipid aggregates (PL) inside of lysosomes of he-patocytes (phospholipidosis). (E) Large areas of lytic necrosis (NE)around blood vessels in fatty liver (arrow). (F) Adenoma (AD) withbasophilic staining properties of altered cancer cells that can be clearlydistinguished from the surrounding liver parenchyma (LP). Magni-fication of 3400.

Fig. 2. Electron-microscopic photographs of characteristic morpho-logical changes of lysosomes during progression of toxipathic lesions.(A) Normal small lysosomes with homogeneous structure. (B) Typicallysosomes in a reversibly altered liver with high induction of CYP450activity that contains fibrillar elements, black lipofuscin granules, andfields of digested ribosomes (arrows). (C) Lysosomes taking up lipiddroplet (arrows) that had accumulated in the cytoplasm. (D) Uptakeand accumulation of phospholipid whorls into a lysosome (arrow),which gives rise to eosinophilic granules in the cytoplasm as seen atthe light-microscopic level (compare Fig. 1D). LY 5 lysosomes; M5 mitochondria; LIP 5 lipid droplets; PL 5 phospholipid whorls.Magnification of 320,000.

These focal alterations are diagnosed as preneoplastic and weredetected with only 1.8% frequency in the reference area andwith 18% frequency at the most contaminated site.

Basophilic foci are among the most commonly occurringpreneoplastic lesions found in flounder in the North Sea. Clearcell foci consist of hepatocytes with vacuolated cytoplasm,mostly due to large lipid vacuoles, and are one of the mostcommon toxicopathic lesions encountered after basophilicfoci. More difficult to detect because of their high degree ofsimilarity to normal hepatocytes are eosinophilic foci, whilemixed cell foci may only occasionally be encountered and areplaced in the category of the predominant cell staining type.For lysosomal changes, we measured the parameters in thesurrounding parenchyma around the foci because of the al-ready-mentioned problem of cutting serial sections of thesesmall lesions.

Benign hepatocellular neoplasms (adenomas) may be rec-ognized by a set of morphological criteria that apply to allvariants and are described in detail elsewhere [32]. Dominantfeatures are compression of the surrounding tissues, a relativeabsence of exocrine pancreatic tissue and bile ducts, and fewmelanomacrophage centers (Fig. 1E).

Malignant hepatocellular neoplasms (carcinomas) are char-acterized by several key morphological and cellular featuresthat generally relate to their aggressiveness and lack of cleardifferentiation, including nuclear and cellular pleomorphism,loss of cellular polarity, anaplasia, invasion of adjacent tissue,irregular borders of the lesion, and obvious satellites with var-iable shape. Serial sectioning of larger nodules frequentlyshowed transitional stages from benign to malignant carci-nomas according to morphological criteria, invasive fronts, andsatellites. Only 1% of the flounder showed benign neoplasmsat the least polluted site compared with 9% of benign andmalignant hepatocellular tumors in the Elbe estuary.

Storage disorders of lysosomes

Normal flounder liver described as healthy contained onlya few small lipid droplets and stored glycogen in the cytoplasmas a reserve substance. Normally, lysosomes are small, havea homogeneous texture (Figs. 2A and 3A and B), and arelocated close to the Golgi region at the biliary poles of he-patocytes.

With the initial signs of toxic injury, an increase of small

2438 Environ. Toxicol. Chem. 21, 2002 A. Kohler et al.

Fig. 3. Early and progressive liver injury: Histochemical determination of morphological and functional responses of lysosomes in serial tissuesections. (A) Unsaturated neutral lipids in hepatocytes as visualized by Oil Red O. (B) Increase in size of lysosomes calculated as volume density.(C) Number of lysosomes per unit area of liver parenchyma as numerical density (except macrophages). (D) Membrane stability measured astime intervals needed for acid labilization of the lysosomal membrane. Long intervals reflect high stability and short intervals reflect reducedstability, which is indicative of impaired membrane function.

lipid droplets in the cytoplasm occurred. In reversibly injuredlivers, we observed electron microscopically that lysosomestake up large numbers of ribosomes (Fig. 2B). We also notedthat most of the lysosomes of hepatocytes engulfed and ac-cumulated lipid (Fig. 2C). A progressive, highly significantaccumulation of lipid, histochemically determined as unsatu-rated neutral lipids, was associated with irreversible liver de-generation (Figs. 3A and 4A). During the progression fromtoxipathic to neoplastic lesion type, heteromorphous materialfrom damaged cell organelles and phospholipid-like whorlsaccumulated in degenerated livers and were also engulfed bylysosomes (Fig. 2D). Intralysosomal accumulation of neutrallipids was clearly demonstrated by histochemistry of neutrallipids and lysosomal enzymes in serial sections (Fig. 4).

In surrounding parenchyma of preneoplastic lesions, ade-nomas, and carcinomas, the intralysosomal lipid was also in-creased but showed a lower variability in the range of highlipid levels (Fig. 5A). In contrast, lysosomes of cancer cellscontained only very low amounts of unsaturated neutral lipids(Fig. 5A). Ultrastructurally, they were homogeneously electrondense and were situated close to the Golgi stacks of the cancercells representing primary lysosomes. Additionally, in baso-philic tumor cells, large heterophagic vacuoles were observed,whereas heterophagic activity was directed toward extrafocalnormal hepatocytes at the periphery of the cancer.

Size and number of lysosomes

With accumulation of neutral lipids, phospholipids, anddamaged cell organelles inside the lysosomes, the size of thelysosomes increased only moderately (Figs. 3B and 4B). Sig-nificant differences were statistically analyzed only betweenhealthy livers and degenerated livers without and with pre-neoplastic foci. Concomitantly, we observed that, with theenlargement of lysosomes, their numbers increased in non-neoplastic degenerated livers (Fig. 3C). Yet in extrafocal pa-renchyma around preneoplastic lesions, a decrease in the num-ber of lysosomes occurred while volume density stayed high;this is indicative of fusion events of lysosomes in preneoplasticlivers. In reversibly and irreversibly injured livers, accumu-lation of lipoid materials was negatively correlated with mi-crosomal and total protein of the liver (Spearman’s rank cor-relation, p , 1%; data not shown).

In contrast, lysosomes of cancer cells were significantlyaugmented compared with extratumoral liver cells and weresignificantly enlarged, as reflected by high values of volumedensity (Fig. 5B and C).

Membrane stability of hepatocellular lysosomes

Livers diagnosed as healthy showed high stability of themembranes of hepatocellular lysosomes, as reflected by longdestabilization intervals of up to 35 min. With the onset of

Lysosomes as prognostic biomarkers in fish Environ. Toxicol. Chem. 21, 2002 2439

Fig. 4. (A) The lysosomal marker enzyme (n-acetyl hexosamidase)visualizes small lysosomes in healthy livers and (B) very low amountsof neutral lipids (arrows). Considerably enlarged lysosomes, as in-dicated by circles, with peripheral enzyme localization (C) containlarge amounts of neutral lipids (D). We selected an extreme case todemonstrate the principle of intralysosomal lipid accumulation. Mag-nification of 3400.

toxic injury, loss of integrity of the lysosomal membrane oc-curred simultaneously, as indicated by a shortened labilizationtime of less than 10 min (Fig. 3D). Membrane stability oflysosomes remained reduced independently of the further pro-gress of liver injury. In extrafocal liver tissue of neoplasticlesions, the lysosomal stability was low, too, as expected. Yetin benign and malignant cancer cells, lysosomes displayedvery high membrane stability and an acid labilization intervalof up to 35 min (median) was needed before the substratecould enter the lysosomal interior (Fig. 5D). These observa-tions show that lysosomes of tumor cell are unimpaired bytoxins and that their membranes have unaffected functionalintegrity.

Contaminant concentrations and liver parameters

Parallel analysis of contaminant concentrations evidencedclear correlations of levels of foreign compounds with toxicliver injury and lysosomal changes in livers. In general, wefound significant positive correlations between the accumu-lation of the majority of PCB congeners and other organo-chlorines and lipid accumulation, negative correlations withthe integrity of the lysosomal membrane, and ultimately, apositive correlation with the degree of liver damage diagnosedhistologically (Table 1).

The highest concentrations of HCH isomers and the PCBcongeners 118, 138, 170 in the liver, e.g., always coincidedwith very short intervals of acid destabilization of the lyso-somal membrane, i.e., between 0 and 10 min (Fig. 6). Low

concentrations of contaminants in livers always coincided withthe longest destabilization intervals of lysosomes, i.e., morethan 15 min, significantly different from highly contaminatedlivers (p , 0.01). High variability of contaminant levels wasfound in livers with a medium labilization time, i.e., between10 and 15 min.

With respect to heavy metals, membrane stability of ly-sosomes and histopathology of the liver were correlated onlywith the amount of cadmium, while links to potential effectsof other metals could not be identified statistically. Further-more, cadmium and lead levels were correlated negatively withthe amount of unsaturated neutral lipids (Table 1) and totalliver lipid and correlated positively with the protein content.

DISCUSSION

In this multidisciplinary study, loss of lysosomal membraneintegrity, changes in lysosomal morphology, accumulation ofvarious types of lipids inside the lysosomes, and histopathol-ogy were significantly correlated with the accumulation oforganochlorines in livers of flounder (P. flesusu (L.)). Earlierstudies paid attention only to a limited range of parametersand determined lysosomal membrane damage alone in relationto histopathology of the dab liver; both indices showed as-sociations with the pollution levels measured in water andsediments along a North Sea transect during the BremerhavenInstitute of Ocean Sciences/International Council for the Ex-ploration of the Sea workshop [16]. In other field studies withfish species of the Mediterranean Sea, the Red Sea, and theNorth Sea, liver cryostat sections and isolated hepatocytesshowed relationships between reduced lysosomal membranestability and increased concentrations of organochlorines inmuscle tissue [18,19,50]. Nevertheless, these studies unam-biguously indicated the value of the determination of lyso-somal membrane stability as a biomarker of the effects ofpollutants. For example, single acute discharges of DDT andPCBs in the early spring of 1996 and remobilization of con-taminants after 1998 from riverbed deepening of the RiverElbe became obvious and were clearly identified by reducedlysosomal membrane stability in flounder liver [18]. Advan-tageously, lysosomal membrane stability in flatfish liver ro-bustly reflects different contamination scenarios independentlyof the time of the year, including the periods of reproduction[15]. In contrast, other important biomarkers such as CYP450activity decrease dramatically in females during reproduction[51].

Numerous studies in invertebrates over the past 25 yearshave proven that lysosomes of the digestive gland accumulateand respond to various classes of pollutants, including organ-ochlorines, PCBs, polycyclic aromatic hydrocarbons (PAHs),metals such as Cr, Cu, Pb, and Zn, and organometals such astri-n-butyltin. For example, the effects of the heavy metals Cu,Cd, HG, and Zn and radionucleotides (uranium 238, plutonium239, and americium 241) on lysosomes (e.g., accumulation ofneutral lipids, reduced membrane stability, and augmented li-pofuscin) were validated as biomarkers for pollutants versusother biomarkers of general stress, such as heat, salinity, andultraviolet light, in field and laboratory studies in various spe-cies of invertebrates [52–57].

Classes of organic compounds comprising PAHs and or-ganochlorines exerted clear detrimental effects on the functionof lysosomes in invertebrate digestive glands during short- andlong-term exposure in caging and transplantation experimentsas well as in field studies [58–64]. Effects of their combination

2440 Environ. Toxicol. Chem. 21, 2002 A. Kohler et al.

Fig. 5. Liver tumors (adenoma/carcinoma) and extralesional liver parenchyma: Histochemical determination of morphological and functionalresponses of lysosomes in serial tissue sections. (A) Unsaturated neutral lipids as demonstrated by Oil Red O are significantly lower in proliferatingtumor cells. Volume density (B) and numerical density of lysosomes (C) were significantly increased in tumors. The membrane stability (D) oflysosomes in tumor cells was significantly higher even than that of hepatocytes of normal, healthy flounder (compare Fig. 3D).

with heavy metals (e.g., phenathrene, Cu, and Cd) on mem-brane integrity and lysosomal lipofuscin storage have beendescribed in Littorina littorea, Perna viridis, Crassostrea vir-ginica, and Mytilus edulis [65,66].

Much less information is available on lysosomal responsesin the liver of teleost fish and marine mammals and is mainlybased on morphological criteria at the light- and electron-mi-croscopic levels. Prolonged exposure to endosulfan, e.g., in-duced an increase in lysosomal size and heterology in liver oftrout and eel [67], while exposure to Hg alone induced ex-cessive accumulation of lipofuscin and amorphous material indolphin liver associated with liver anomalies [68].

In the present study, an increase in lipid was clearly cor-related with the accumulation of organochlorines and PCBs inthe same livers. Enhanced autophagy of excessive cytoplasmiclipids (lipidosis) and phospholipids (toxic phospholipidosis)by lysosomes lead to accumulation and storage of lipid-boundorganic toxins inside lysosomes [13,58,69–71]. Lipid accu-mulation in the cytoplasm and lysosomes, especially in teleostlivers of the glycogen-storing type, is regarded as a valuableindicator for early toxic injury of liver and digestive glands[28,42,71–73]. We found that heavy metal concentrations in

flounder liver were negatively correlated with the accumula-tion of lipid during progressive liver lesions associated withloss of protein content. This suggests that binding sites formetals in flounder hepatocytes were reduced. As demonstratedin the North Sea flatfish Limanda limanda by Soffker [48],lipids stored in flatfish liver are the sites of accumulation forcertain organochlorines and PCBs, which are partly transferredto the gonads during the reproductive cycle.

The fact that we observed an increase both in size andnumber of lysosomes in degenerating livers indicates that pro-liferation of lysosomes is stimulated by injured cell organellesand lipids storing chemicals in order to cope with the toxicinsult. Similar observations were described in fish after ex-perimental exposure to benzo[a]pyrene and in field studies[74,75]. Only in livers with preneoplastic foci did we observea decrease in number of lysosomes accompanied by an increasein volume, which is indicative of fusion events between ly-sosomes, a frequently described stress phenomenon in musselsinduced by augmented degradative demands [58,73,76]. Ar-nold et al. [67] interpreted the increase of lysosomal size inrainbow trout liver after prolonged endosulfan exposure as acharacteristic of chronic effects, which is in accordance with

Lysosomes as prognostic biomarkers in fish Environ. Toxicol. Chem. 21, 2002 2441

Table 1. Correlations between concentrations of xenobiotics (mg/kgwet wt of liver) and cell and tissue responses measured in identicallivers as indicated by Spearman’s rank correlation coefficient p , 0.01

(indicated by italics); standard p , 0.05 level of significance)

Xenobioticsa Biological responses

PCB congeners 28, 101, 118,128, 138, 170, 177, 183, 187

HCH isomersHexachlorobenzene

Unsaturated neutral lipidsPositive

Cadmium, lead Negative

PCB congeners 101, 118, 128,138, 149, 153, 170, 183,187, 194

HCH isomersCadmium

Lysosomal membrane stabilityNegative

PCB congeners 101, 118, 128,138, 149, 151, 153, 170,183, 187

b-HCHDDD, DDEOctachlorostyrene

Progression of liver injuryPositive

a PCB 5 polychlorinated biphenyl; HCH 5 hexachlorcyclohexane;DDD 5 dichlorodiphenyldichloroethane; DDE 5 dichlorodiphen-yldichloroethylene.

Fig. 6. Examples of polychlorinated biphenyl congeners and organ-ochlorine concentrations in liver of flounder tested histochemicallyfor lysosomal membrane stability. Impaired membrane stability con-sistently coincided with high levels of the polychlorinated biphenylsCB 118, 138, 170 and the isomers a-hexachlorcyclohexane (HCH0and b-HCH as well as total lipid content of liver. High stability ofthe lysosomal membrane (as indicated by long destabilization inter-vals) were always associated with low concentrations of lipophiliccompounds. Medium stability of lysosomes were related to concen-trations of chemicals with high variability, which is interpreted asdifferences in individual capacity of fish liver to compensate for toxicinjury.

the fact that the development of preneoplastic and neoplasticlesions is a long-term process [77].

The underlying mechanisms of toxic injury of the lysosomalcompartment have been most extensively studied in inverte-brates. In mussels and scallops (M. edulis and Adamussiumcolbecki, respectively), increased protein catabolism was ob-served to be closely linked with decreased lysosomal mem-brane stability [56,66,78]. We observed accumulation of ri-bosomes in lysosomes and an increase in size at the ultrastruc-tural level that was associated with a decrease of total liverprotein in reversibly injured livers (Wahl et al. [17]). Theseobservations were clearly related to increased protein catab-olism, while lysosomal membrane integrity was reduced. Onthe basis of earlier results, a new hypothesis for the mecha-nisms of lysosomal membrane damage can be formulated forliver of fish. In reversibly injured livers, leakage of lysosomalmembrane coincided with induction of biotransformation en-zymes of the CYP450 biotransformation system [79], whichare known to produce reactive oxygen species and reactiveoxygen metabolites and consequently induce lipid peroxida-tion of cell membranes, including those of lysosomes [80,81].Winston et al. [82,83] found that formation of reactive oxygenspecies also occurs inside lysosomes by interaction of ingestedxenobiotics (copper, PAH), whereas PAHs are attacked by al-ready-present metal ions inside lysosomes, resulting in theproduction of anthraquinones, which undergo redox cycling.Brunk et al. [84] identified intralysosomal iron-catalyzed ox-idative reactions associated with membrane damage and leak-age of hydrolytic enzymes. Additionally, Regoli et al. [56]stated that, in metal-exposed Antarctic scallop, a dramatic lossof cytosolic antioxidative enzymes, glutathione, and catalasewas associated with reduced lysosomal membrane stability.Thus, oxyradical attack of the lysosomal membrane might notoccur only from the cytoplasmic side but from the interior ofthe lysosomes as well.

With progressive liver lesions, the malfunction of the ly-sosomal membrane of flounder liver was associated with de-

creased degradation of ingested material. Undigestible mate-rial, accumulated in secondary lysosomes, is generally un-loaded from hepatocytes and macrophages in a process ofcellular defecation by the discharge of residual bodies [85], aprocess we only observed after transfer of flounder into cleanwater during regeneration experiments [86]. Storage of sub-stances in lysosomes occurs either because the substances arenot digestible or because of enzyme deficiencies. To enablelysosomal enzymes to work, an acid environment with a pHof approximately 4.8 has to be maintained inside the lysosomeby a hydrogen-ion pump [87,88]. Internal alkalinization oflysosomes was observed by proton carriers (uncouplers) suchas organometals, e.g., tributyl lead [89], associated with thedecreased activity of pH-dependent hydrolases.

In the present study, damage of the lysosomal membraneof hepatocytes was already evident in livers displaying earlytypes of lesions regarded as reversible [86]. In contrast withthis rapid loss of membrane integrity of lysosomes, lipid ac-cumulation and the increase in size of lysosomes were moreor less continuous processes, with progression of toxic injuryin livers without and with preneoplastic lesions and cancers.

2442 Environ. Toxicol. Chem. 21, 2002 A. Kohler et al.

Therefore, membrane stability and release of lysosomal en-zymes into the cytoplasm can be regarded as a very earlybiomarker that has prognostic value for later stages of liverdegeneration and cell death, as has already been postulated byKerr [85]. On the basis of the findings of Regoli [90] duringchronic heavy-metal exposure and of Lowe et al. [91] for oilcompounds, it can be stated that reduced lysosomal membranestability is not a transient phenomenon. Lysosomal membranedestabilization persisted after transfer of mussels into cleanenvironments for up to four weeks, and we can expect similarresponses of lysosomes in fish liver.

Additionally, a reduced potential to recover with increasingage, as was observed by Hole et al. [92] in marine mussels,may contribute to persistence of impaired lysosomal functionin fish liver, which promotes progression of cellular degen-eration, consequently resulting in cell death of normal hepa-tocytes. These events may give rise to better adapted preneo-plastic hepatocytes, diagnosed as foci of altered hepatocyteson the basis of morphological criteria. In contrast with thetoxically injured extrafocal liver parenchyma, lysosomes ofneoplastic hepatocytes were numerous and of very high mem-brane stability, as can be expected from proliferating cells.Therefore, lysosomes of cancer cells are highly capable ofenclosing and recycling macromolecules, including xenobi-otics.

In earlier studies, we measured a metabolic shift of detox-ification enzymes in preneoplastic foci and benign and malig-nant hepatocellular cancers of flounder. These changes wererelated to increased PCNA-labeling, proliferating cell nuclearand increased activity of G6PDH (glucose-6-phosphate de-hydrogenase), the key enzyme of the pentose phosphate shunt,which is involved in production of reduced nicotinamide-ad-enine-dinucleotidephosphate as a reduction equivalent for bio-transformation and oxyradical scavenging and pentoses fornucleic acid synthesis. The CYP450 protein was reduced inaltered hepatocytes and cancers, and thus, formation of dam-aging oxyradical species in cancer cells was prevented. Inparallel, increases of both glutathione-S transferase and the P-glycoprotein drug efflux pump mediating xenobiotic resistancewere observed. These metabolic changes led to the hypothesisthat populations of hepatocytes forming preneoplastic foci,adenomas, and carcinomas appear to be better adapted againsttoxic insults compared with normal injured hepatocytes andtherefore have a clear growth advantage [23,29].

The rationale for using the model of lysosomal functionversus dysfunction was to examine the mechanisms that occurprior to and during formation of degenerative lesions and dur-ing carcinogenesis in the liver of fish, with the aim of devel-oping rapid assays in monitoring programs for the detectionof biological effects of pollutants. Used in concert with otherbiomarkers, the measurement of lysosomal changes as an indexfor health of liver cells plays a key role for the interpretationof responses of other biomarkers, such as CYP450 induction.Our study showed that lysosomal perturbations have predictivecapability as a diagnostic tool for selected ecologically relevantendpoints, such as cell death and survival of better adaptedhepatocytes, supporting the process of carcinogenesis. In con-trast with the pathologies of mussel hepatopancreas, the liversof fish display a complexity of lesion types similar to themammalian liver. Therefore, it is important to develop screen-ing tests that are easy to apply, that can be substituted forexpertise-dependent histopathological diagnosis, and that areaccurate, sensitive, and rapid.

Acknowledgement—We are grateful to David Lowe and Mike Moore(PML, Plymouth, UK), old friends and colleagues in research, forintroducing us to the histochemical approach to testing lysosomalmembrane integrity and thus helping to achieve the development oflysosomes as biomarkers in fish liver. We also want to thank UweHarms (BFA, Hamburg, Germany) for analyzing the livers of flounderfor heavy metals. The data evaluated for this article were accumulatedthroughout the research projects Fish Diseases in the Wadden Sea andFish Diseases in the North Sea, financed by the Environmental Agency(UBA Berlin, Germany).

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