Marine bottom-dwelling flatfish, such as flounder (Platichthys flesus (L.)), are most affected by liver cancer because they live in close contact with sediments that accumulate hepatotoxic and (pro)carcinogenic compounds and carcinogenesis-promoting agents. The Elbe is one of the most polluted European rivers and has a major impact on the German Wadden Sea. Tumour frequencies up to 70% have been found macroscopically in female flounder at the most polluted site in the Elbe estuary. Key events of carcinogenesis in flounder liver have been described in detail elsewhere. These events start off with the appearance of foci of initiated eosinophilic hepatocytes and their subsequent transition into basophilic foci. The occurrence of a basophilic cell type is a key step in neoplasia because this cell type persists in carcinomas and their invasive protrusions (satellites). Histopathological alterations in flounder liver are identical to those found in field studies and experimental studies of fish and rodents.

Pollution of the Elbe estuary is reflected by elevated levels of polychlorinated biphenyl congeners (PCBs) and other fingerprint organochlorines such as lindane isomers, DDT metabolites, hexachlorobenzene and octachlorostyrene in flounder liver in comparison to individuals caught in a less polluted reference site. Elevated levels of the heavy metals mercury and chromium have been found in muscle tissue. The majority of these compounds have been identified as cytotoxins and (pro)carcinogens in rodent models. Levels of organochlorines and heavy metals in liver and muscle tissue of flounder in the Elbe estuary have been shown to be directly related to age and duration of stay.

Interestingly, liver tumours of flounder that have been investigated in the present study did not show specific mutations that are frequently found in mammalian cancers such as mutations in exons 5-8 of the tumour-suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. Therefore, we hypothesised that a xenobiotic-resistant phenotype develops in (pre)malignant cancer cells by metabolic adaptations during chronic exposure to (pro)carcinogens in the polluted habitat of flounder. To study this hypothesis, we determined amounts, localisation patterns and activity of a series of proteins that are relevant for the protection of cells against xenobiotics and their elimination.

Mechanisms of intracellular protection against toxic injury, such as biotransformation of xenobiotics to water-soluble metabolites by phase I enzymes, scavenging of radical metabolites and conjugation to excretable molecules by phase II enzymes as well as lysosomal accumulation of toxins, have been studied thoroughly in the past in various marine species including flounder. Less attention has been paid to a first line of defence (phase 0 enzymes) that is localised in membranes of cells to prevent accumulation of xenobiotics in cells, the transmembrane P-glycoproteins (P-gps) and related transporters. P-gps are ATP-dependent drug efflux pumps that transport a wide variety of chemicals over membranes and mediate the phenomenon of “multidrug resistance” or “multixenobiotic resistance” (MDR/MXR). P-gps are members of the ABC transporter gene family such as the bile salt export pump (BSEP) and multidrug resistance-related protein (MRP) that contribute to MDR. MDR, that render cancer cells resistant to chemotherapy, is one of the major obstacles in the treatment of cancer. Transporters of drugs or xenobiotics have been found as well in aquatic invertebrates and fish (flatfish, blenny, mummichog; Bard, 2000). A large number of anthropogenic pollutants such as polycyclic aromatic hydrocarbons (PAHs), PCBs and DDT and their metabolites can either induce or inhibit expression of these transporters in fish, invertebrates and mammals and are referred to as chemosentisisers.

To analyse which metabolic events render hepatocytes resistant against chronic toxicant exposure, we determined expression and localisation patterns of P-gp during early toxic injury in juvenile flounder and progression of liver lesions in adult flounder. Besides, we localised cytochrome P450 (CYP450IAI) which converts lipophilic chemicals into water-soluble compounds (phase I of biotransformation) to facilitate detoxification and excretion via urine, bile and over the gills. However, phase I biotransformation may also promote activation of xenobiotics to reactive radical intermediates that are toxic and even carcinogenic. Furthermore, we analysed expression of glutathione-S-transferase-A (GST-A; phase II of biotransformation) because it binds and transports toxic endobiotics and xenobiotics and conjugated electrophilic compounds including radical metabolites. GST-A overexpression has been described in mammalian tumours and cancer cell lines and is associated with the development of a MDR phenotype. Finally, we localised activity of glucose-6-phosphate dehydrogenase (G6PDH), key enzyme of the pentose-phosphate pathway that generates pentoses and NADPH necessary for biosynthesis and detoxification processes and is elevated in (pre)cancerous lesions in humans, rodents, flounder and other fish species.

European flounder (Platichthys flesus (L.)) is the main target species for biological effects monitoring, for example in European coastal zone management (North Sea, Baltic Sea, Adriatic Sea, Mediterranean Sea) since the species is abundant, bottom dwelling and inhabits shallow waters such as the Wadden Seas and estuaries along salinity gradients. Juvenile flounder immigrate into shallow waters of the Wadden Sea and estuaries after metamorphosis. In the Elbe estuary, they reside in feeding habitats and are chronically exposed to an “Elbe-characteristic” contaminant mixture until the age of 4 years. At this age, their residence is shortly interrupted from January to March to spawn in the southern North Sea from where they return to the feeding grounds. Female flounder (length, 18–40 cm; age classes II–VIII) were caught in the Elbe estuary and in less-contaminated offshore areas, outside the spawning period. Flounder were killed by a blow on the head and a cut through the spine immediately after capture. Pieces of liver (5×5×5 mm³) were dissected and deeply frozen at ?70°C in supercooled hexane and stored at ?80°C. Parallel samples of liver and the various types of lesions were fixed in 4% Baker’s formaldehyde for embedding in methacrylate (Merck, Darmstadt, Germany). For immunoelectron microscopy of P-pg-related transporter proteins, liver blocks (1×1×1 mm³) were fixed in 0.1% glutaraldehyde and 3.7% paraformaldehyde for 2 h.

Diagnostic criteria are summarised in detail in the recommendations of the ICES Special Meeting on the Use of Liver Pathology of Flatfish for Monitoring Biological Effects of Contaminants and in the guidelines of the BEQUALM workshop for future use under the OSPAR Joint Assessment and Monitoring Programme. These criteria include cytoplasmic and nuclear changes as well as morphology of hepatocytes during non-neoplastic liver injury. Reversibility of non-neoplastic liver lesions have been tested by regeneration experiments of flounder using flow through systems and uncontaminated food over a period of 4 weeks and more. Staining properties of foci of altered hepatocytes, hepatocellular adenomas and carcinomas, as well as growth patterns and invasive growth were used to diagnose sequential changes during carcinogenesis.

Altogether, 185 foci of altered hepatocytes, hepatocellular adenomas and carcinomas were diagnosed histopathologically in cryostat sections of 125 livers. Histopathological data were correlated with expression and activity of P-gps, CYP450, GST-A and G6PDH quantified with the use of image analysis in a selection of 5 to 15 cases of each lesion type.