Marine Environmental Research 28 (1989) 417~,24

Cellular Effects of Environmental Contamination in Fish from the River Elbe and the North Sea

Angela K6hler

Biologische Anstalt Helgoland, Zentrale Hamburg, Notkestrasse 31, 2000 Hamburg 52, FRG

ABSTRACT

Results of the tissue and cellular responses (light and electron microscopy, eytochemistry ) of the liver oJflounder (Platichthys flesus L.) from a current interdisciplinary study are presented. Fish were sampled ahmg a pollution gradient from the Elbe estuary towards the northern part of the Wadden Sea coast and the Eider estuary (reference areas). Livers of d(fferent age groups ~?[" flounder (0, L H, III) were investigated in order to relate the degree qf tissue lesions to the 'exposure time' or lifespan the .[ish inhabits the d!ff'erently contaminated areas. The severi O, of pathological liver changes corresponds to the accumulation of contaminants (HCB, PCBs, OCS, d,y-HCH, ttg) and df~ers s~n(l~'cantly with prohmged residence at the sites investigated. Liver ultrastructure revealed evidence o['severe perturbations of the intraeellular organi:ation (disorgani-ation ~71 rough endoplasmic reticulum, lysosomes- lipoJi~scin, lipid aecumulation, de[cots of ~3'toskeleton) to cellular responses indicating the initiation o[ detoxoqcation processes (smooth endoplasmic reticulum, lysosomes) and "normal' compartmentation o['hepatocytes along the pollution gradient investigated. Based on the studies ~[" normal liver structure and its pathological phenomena indicating a k O' role o[ the Ivsosomal O,stem, the O'sosomal stability test (N-aceO'l-hexosaminidase) was applied in order to relate tissue and celhdar pathology to cytochemical indices re[tecting the bioehemistry t?['the in/ured tissue. First results revealed a highly significant negative correlation between the stabili O, ~['the O~sosomal membrane to the degree ~?[" liver lesions. Furthermore, the initial results obtained from the mouth ~?[the Elbe estua O, and a northern r~[brence area in the Wadden Sea in[~rred sign([icant d(fferences with respect to lvsosomal stabilit v.

417

Marine Environ. Res. 0141-1136/90/$03.50 1990 Elsevier Science Publishers Ltd. England. Printed in Great Britain

418 Ange~ K6h#r

The recent interdisciplinary study in the German Wadden Sea (North Sea) and in the adjacent estuaries (Eider, Elbe, Weser, Ems) was intended to link together different methods of environmental research to test their sensitivity to early responses of the fish liver to anthropogenic stressors. These methods include the stepwise identification of pollution-induced changes on the organ, cellular and biochemical level by gross and microscopic (light and transmission electron) morphology, cytochemistry (lysosomal stability), biochemistry (mixed-function oxidase (MFO) activity) and chemical analyses of representative contaminants.

The Elbe is one of the most polluted rivers in Europe and is characterized by a decreasing gradient of contaminant levels in water and sediments towards the mouth of the estuary. ~ -4 Selected data on the cellular effects observed in the liver of flounder caught along a pollution gradient from the inner bounderies of the Elbe estuary (station 1) to the mouth (station 2), northward in reference areas at the Wadden Sea coast (station 3) and in the Eider estuary (station 4) are reported. In order to detect early sublethal responses and their progression towards severe liver lesions in relation to the 'exposure time,' different age groups of flounder (0, I, II, III) were investigated. Exclusively, flounder before the first sexual maturity were selected in Order to exclude cellular and biochemical effects in the liver during the spawning cycle. Pieces of liver tissue were processed for light and electron microscopy and for the lysosomal stability test (N-acetyl-fl- hexosaminidaseS). All data on contaminant concentrations (polychlorinated biphenyls, PCBs, hexachlorobenzene, HCB, d,y-HCH, OCS, Hg) referred to in this paper were already published. 6

Prolonged duration of stay in the highly polluted part of the Elbe estuary, near the city of Hamburg (station 1) was associated with dramatic increase of liver damage including steatosis, extensive necrosis and pre-neoplastic changes (megalocytic hepatosis, eosinophilic and hyperplastic foci) in 60 70% of the older age groups (Table 1). Compared to normal ultrastructure (Fig. la) these lesions included extensive disorganization of the rough endoplasmic reticulum (RER), proliferation of a smooth membrane system associated with lipid droplets, atrophy of Golgi complexes and inhibition of lysosomal formation, apparent increase of lipofuscin granules, mitochondrial degeneration and cytoskeletal per- turbations (microtubule formation in the RER lumina) (Fig. lb, d, g and h). The extension of pathological changes with age was accompanied by an age- dependent accumulation of hepatotoxic substances in muscle tissue (fresh weight) up to maximal levels of 3100#g/kg PCBs, 1907/~g/kg HCB and 905/tg/kg Hg.

The flounder population permanently inhabiting the less polluted area near the mouth of the Elbe estuary (station 2) showed only minor changes

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Cellular effects ~1 contamination in fish 421

reflecting the morphological correlates to detoxification processes (increase of RER, proliferation of Golgi complexes, increased number of lysosomes) (Fig. lc and e). No further accumulation of contaminants above the age of 1 year were seen. Seasonal observations at this station revealed that in late summer a second type of liver lesion (cf. Table 1/station 2) occurred, closely resembling the tissue and cell phenomena characteristic for station 1. Besides, clear signs of initial reorganization and detoxification processes could be identified: an extensive development of tubular smooth endoplasmic reticulum (SER) and reorganization of RER as parallel stacks (Fig. l f), the dissolution of microtubular structures (paracrystals), enlarge- ment of Golgi complexes and increase of lysosomes. Nearly identical patterns were observed during the experimental regeneration of the liver of tlounder caught at the highly polluted area of the Elbe estuary (station 1). The regeneration process during the contaminant-free maintenance was dependent on the elimination of PCBs and related lipophilic substances and the binding of Hg in a non-toxic form, presumably by metal-binding proteins and lysosomes. 7

Ranges of contaminant levels of flounder caught at station 2 only in late summer and autumn closely resembled annual ranges of station 1 flounder. Dramatic oxygen deficiencies in the region near Hamburg probably led to downstream migration towards the mouth of the estuary, thereby mixing of two differently contaminated flounder populations occurred. Livers of flounder caught in the reference area (station 4) showed only minor changes (Table 1). Hepatocytes displayed a characteristic compartmentation of mitochondria, Golgi complexes with few small lysosomes and an owd nucleus surrounded by parallel stacks of RER, and of large fields of reserve

Fig. I. Characteristic ultrastructura] findings (total N-60) . (a) Normal liver cells of flounder caught in the reference area (station4). (b) Hypertrophied hepatocytes tl~ surrounded by a network of dark shrunken cells H, in flounder liver caught in the highly polluted part of the Elbe estuary (station 1). Note the disorganization of RER, large lipid droplets, lipofuscin granules and nuclear indentations and pycnosis. (c) Extensive proliferation of RER and increased n umber of lysosomes at station 2. (d) Damaged lysosomc transl\~rming into a lipofuscm granule at a large lipid droplet tslation 1). (c) Autophagic 13sosome digesting mitochondria and other cellular debris, characteristic Ik)r station 2. If) Reorganization of parallel stacks of RER from tubular and vesicular ER in liver cells of highly contaminated flounder migrated downstream to the less polluted river mouth. (g) Smooth mcmbranes penetrating large lipid droplet {station 1). (h) Polymerization of tube-like paracrystals at polysomes {insert) and aggregration in the lumina of the RER, morphologically identitied as enlarged microtubules (station 1). RER, rough endoplasmic reticulum: ER, tubular and vesicular endoplasmic reticulum, N. nuclei: M, mitochondria Gly. glycogen; L, lipid: Ly, lysosome" LF, lipofuscm: AV, autophagic vacuole: MAT, macrotubule: SD, space of Disse: BD, bile duct: BC, bile canaliculi: H ~. hypertrophied liver

cell: H: shrunken liver cell.

422 Angela Kdhler

substances (mainly c~-glycogenl (Fig. la). Contaminant concentrations reflected natural background levels for Hg and only slightly enhanced levels for PCBs and related lipophilic substances.

Damage to the lysosomal membrane or overloading of the storage capacity by a variety of toxic compounds leads to increased fragility of the lysosomal membrane in mammals and invertebrates with subsequent release of degrading enzyme to the cytosol and catabolism of cell components leading to cell death. 1'5"8"9 Our findings in normal and injured flounder livers suggest a role for lysosomes in responses to anthropogenic contaminants. The lysosomal stability test, established for stress responses in marine investebrates, 1 was applied to the flounder liver. Degree of liver lesions and lysosomal stability revealed a highly significant negative correlation (Spearman's test, r = -0"716 (Fig. 2a). In normal and slightly affected livers (lesion degree 1 and 2) the mean labilization period was 15-18 rain. Liver lesions above degree 3 were accompanied by drastically reduced mean labilization periods (3-5 rain).

The initial results on lysosomal stability of flounder liver caught at station 2 and station 3 showed significant differences (P < 0'05) (Fig. 2b and c). In the Elbe estuary, short lysosomal destabilization periods of flounder

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Fig. 2. (a) Mean lysosomal labilization period at the different degrees of histopathological liver lesions. The labilization period is the time of pretreatment (citrate buffer pH 4"5) required to destabilize the lysosome membranes completely, measurable as maximum staining intensity for the enzyme assayed (N-acetyl-fl-hexosaminidase) by microscopic photometry or

light microscopic assessment. N - 31.

Cellular ely'ects c~f contamination in Jish 423

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flounder caught in mouth of the Elbe estuary (station 2, N -- 25) and (c) of flounder sampled at a Wadden Sea station, which is halfway to the reference area (station 4, .¥ = 25).

liver reflected serious damage of the lysosomal membranes. In contrast, in the Wadden Sea (station 3) labilization periods indicated a significantly higher lysosomal stability. The comparison of the histograms of both stations indicated that the group of lower labilization values at the reference (station 3) closely fits the distribution of variables observed in the Elbe estuary (station 2). Presumably, highly affected flounder from the Elbe estuary emigrate also to the Wadden Sea area. Preliminary restdts on the MFO activity and on chemical analyses of contaminants support this hypothesis (unpublished data).

The integrity of the lysosomal membrane in fish liver tested by the stability test may sensitively reflect breakdown of the adaptive capacity of liver cells leading to damage of cell function and irreversible pathological

424 Angela Kiihler

alteration. Hence the 'lysosomal labilization test' could be used as a sensitive and highly practicable tool for the future monitoring of fish populations.

A C K N O W L E D G E M E N T

This research project is funded by the Federal Environmental Agency.

R E F E R E N C E S

1. Moore, M. N., Mar. Ecol. Progr. Ser., 46 (1988) 81- 9. 2. Arge Elbe., Schwermetalldaten der Elbe. Wassergfitestelle Hamburg, 1980,

66 pp. 3. Arge Elbe., Chlorierte Kohlenwasserstoffe --Daten der Elbe. Wassergiitestelle

Hamburg, 1982, 51 pp. 4. Arge Elbe., Wassergfitedaten der Elbe. Wassergiitestelle Hamburg, 1983 88,

79 pp. 5. Moore, M. N. & Lowe, D. M., In Effi, cts <~/Stress and Pollution on Mar#w

Animals, ed. B. L. Bayne et aL Praeger Publishers, New York, 1985, pp. 190 204. 6. K6hler, A., Harms, U. & Luckas, B., HelgolOnder Meeresunters, 40 (1986)

431 4O. 7. K6hler, A., Aquat. Toxicol., 14 (1989) 203 32. 8. Allison, A. C., In Lysosomes in Biology and Pathology, Vol. 2, eds J. T. Dingle &

H. B. Fell. North Holland Elsevier, Amsterdam, New York, 1975, pp. 178 204. 9. Slater, T. F., In Lysosomes in Biology and Pathology, Vol. 1, ed. J. T. Dingle &

H. B. Fell. North Holland Elsevier, Amsterdam, New York, 1979, pp. 467 92.