Camp. Biochem. Physiol. Vol. IOOC, No. l/2, Pp. 123-127, 1991 Printed in Great Britain

0306~4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc

LYSOSOMAL PERTURBATIONS IN FISH LIVER AS INDICATORS FOR TOXIC EFFECTS OF

ENVIRONMENTAL POLLUTION

ANGELA K~HLER

Biologische Anstalt Helgoland/Zentrale Notkestrasse 3 1, 2000 Hamburg 52, F.R.G.

(Received 1 October 1990)

Abstract-l. Lysomones play a key role in liver injury in fish caused by organic and inorganic xenobiotics. The lysosomal stability test was transferred to fish liver with the aim of testing responsive and practicable methods for biological-effects monitoring.

2. A two-step response of lysosomes in fish liver could be discerned, reflected by the activity (number and size of lysosomes) and the injury (membrane destabilisation) of the lysosomal detoxifying system.

3. Significant differences, with respect to lysosomal enlargement, membrane stability and pathological lipid accumulation, were found along a pollution gradient throughout the year.

4. The lysosomal tests clearly reflect the breakdown of the adaptive capacity of the fish liver to toxic injury. Therefore, a test battery measuring lysosomal perturbations should be recommended for the biological-effects monitoring.

INTRODUCTION

Since the beginning of this decade the public has become increasingly alarmed by the putative biologi- cal consequences of the steadily increasing input of a wide range of toxic chemicals into the marine and estuarine environment. The Joint Monitoring Group (12th Meeting, 1987) stated that there is serious lack in appropriate methods for monitoring the health of marine organisms. Based on this background the Federal Environmen,tal Agency (Umweltbundesamt/ Berlin) initiated an interdisciplinary research project Fish Diseases in the Wadden Sea, in which biological and chemical methods were simultaneously applied to flounder (Plutichthys flesus L.) along the German Wadden Sea coast, including the adjacent estuaries. The aim of the present project is to test and develop practicable monitoring techniques and to deliver guidelines for biological-effects monitoring.

As in mammals, the fish liver is the central organ for the accumulation and detoxification of organic and inorganic contaminants. Earlier ultrastructural studies in flounder liver evidenced severe alterations of the lysosomal system in relation to the contami- nant burden (KBhler et al., 1986, Kiihler, 1989a, 1990). Lysosomes are able to accumulate and se- quester a wide range of both organic and inorganic compounds as well (Allison, 1969; Moore, 1985). Therefore, tests for measuring lysosomal pertur- bations were applied to flounder liver. Cell and tissue pathologies were diagnosed in parallel in order to test the value of the cytochemical measurements in predicting the extent of liver lesions from revers- ible to degenerative, preneoplastic and neoplastic abnormalities.

MATERIALS AND METHODS

Female flounder (Platichrhysflesus L.), between 17-25 cm total length, were sampled at nine areas in the German

Wadden Sea and the estuaries of the Eider, Elbe and Weser every three months during 1988 and 1989. Representative data of the present study along a pollution gradient were selected for this paper. Seasonal variations of lysosomal parameters were investigated at two stations in the Elbe estuary (Station I), and in a less contaminated reference area (Station 2) every two months during 1988 and 1989. Flounder were caught using the research vessel Uthiim (Biologische Anstalt Helgoland) in 30-min hawls with a dragnet (70mm mesh width) and kept in aerated aquaria immediately after capture. The fish were immediately dis- sected on board and pieces of liver (5 mm) deep frozen in n -hexane (- 70°C) supercooled in liquid nitrogen for cyto- chemistry and stored on dry ice. Serial sections cut below -25°C with a Reichert-Jung cryostat were treated accord- ing to Bitensky er al. (1973) and Moore (1976) for the determination of lysosomal stability. For fish liver the acid labilisation measurements, intervals from 0, 2, 5, 10, 15 up to 35 min have to be extended up to 45 min. The acid hydrolases P-N-acetylhexosamidase, arylsulphatase and p-glucuronidase were used. In order to test the applica- bility of this method to other potential monitoring species, the lysosomal stability test was also successfully applied to the livers of dab (Limandu limandu L.) and plaice (Pleuronectes platessa L.). For the determination of the lysosomal labilisation period in fish liver, the lysosomal enzyme /I-N-acetylhexosamidase and its substrate (naph- thol AS-BI-N-acetyl-p-D-glucosamide/Sigma) delivered the most intensive reaction product.

The membrane destabilisation time was determined as the period of acid labilisation (0, 2, 5, 10 to 45 min) required to fully destabilise the lysosomal membrane, so that the sub- strate could penetrate into the lysosome. The breakdown of membrane stability was indicated by the first peak (maxi- mum staining intensity) of the reaction product between the substrate and the lysosomal enzyme. Maximum staining intensity was microscopically assessed by four readings in equal segments of the tissue sections according to the recommendations by Moore et al. (1978). The readings were repeated in random samples with a Zeiss microscope densi- tometer in order to check and intercalibrate the assessments.

Unsaturated neutral lipids were stained with Oil Red 0 (Bancroft, 1967) and microscopically determined according to a grading from 0 to 7 which was based on density

CBP IWJI-2-I 123

124 ANGELAK~HLER

measurements of the staining product for unsaturated RESULTS

neutral lipids. Number and size-of lysosomes were morpho- metrically measured with a MOP Kontron Videoplan as numerical density and volume (Weibel er al., 1969). Tissue samples for liver pathology were fixed in Baker formal, embedded in methacrylate and stained with H & E (Harris) and PAS. For electron-microscopy small liver cubes were fixed in 5% glutardialdehyde on ice, postfixed in 2% osmiumtetroxid and embedded in Epon after dehydration. Ultrathin sections were stained with saturated uranyl acet- ate (in 100% methanol, 5 min) and with lead citrate (accord- ing to Reynolds) for 1 f min. For the statistical evaluation of the data only non-parametric distribution-free tests were chosen (Spearman’s rank correlation; Kruskal-Wallis H- Test; Nemenyi). Significances were accepted with a r 95% level of confidences.

The results of lysosomal perturbations in flounder liver caught along a suspected pollution gradient in summer, 1989, are summarized in Fig. l(a-d). Highly significant differences between the three stations were found with respect to lysosomal stability with lowest membrane stability in the Elbe estuary and increasing values towards the northern station 3. A similar northward gradient could be observed with respect to lysosomal enlargement and numerical density.

Electron-microscopic studies confirmed the pro- nounced differences in lysosomal cytochemistry by lysosomal morphology at the three stations. In healthy flounder livers, a few small lysosomes with

1 2 3

d I

1

Llvcr hwoprthology

4-f l-

Fig. 1. Lysosomal responses in the liver of flounder caught along a pollution gradient form the Elbe estuary towards the northern Wadden Sea.

mainly autophagic activity were located near peri- canalicular Golgi complexes (Fig. 3a). These ultra- structural findings were accompanied by a considerable membrane stability of up to 45 min in single individuals. No pathological alterations could be identified in these livers.

In contrast, liver cell lysosomes of flounder caught in the Elbe estuary drastically increased in number and size. There was a considerable accumulation of pigmented material (melanin, lipofuscin), injured cell components and unsaturated neutral lipids (Fig. 3b). A highly significant negative correlation (r = -0.63 to r = -0.80, N = 25) was found between the en- largement of lysosomes and the number of lysosomes at Stations I and 2 (Fig. 1 a,b), indicating that new lysosomal formations were retarded and that lyso- somes displayed an increased rate of fusion. Also, highly significant but positive correlations were calcu- lated for lysosomal size and for pathological accumu- lation of unsaturated neutral lipids (r = +0.68, N = 25), confirming our anticipation that lipid ac- cumulation was intralysosomal. Interestingly, no cor- relation between lysosomal stability, size, number with the lipid content could be statistically identified.

Highly significant correlations were found between lysosomal membrane stability and the degree of histopathological liver lesions (Fig. 3c, d) ranging from normal and minor reversible changes (stages 1 and 2) to necrosis, fibrosis, cytoskeletal changes, megalocytic hepatosis, caryomegaly, lipid accumu- lation (stages 3 and 4) to heavy staetosis, fat necrosis and cirrhosis (stage 5) and eosinophilic, clear cell and basophilic foci (stage 6).

The observation of the seasonal variations of lyso- somal stability at two sites (Stations 1 and 2) indi- cated decreased lysosomal membrane stability during winter. This fact coincided with the appearance of maturing female flounder in the catches from October until March. Therefore, the seasonal data were sorted according to the activity and rest of gonadal growth and vitellogenesis. During the two years of investi- gation, significant differences could be identified stat- istically between the phases of activity and rest in vitellogenesis, with respect to lysosomal stability at both stations (Fig. 2). Nevertheless, the differences in lysosomal stability between these two stations

20

1 PHASES OF VITELLOGENESIS

Lysosomal perturbations in fish liver 125

remained statistically significant, independent of the phases of vitellogenic activity.

Electron-microscopic studies revealed the partici- pation of the lysosomal compartment in the processes of vitellogenesis in liver. Lysosomal digestive activity was directed towards lipid degradation and phospho- lipoprotein transport (Fig. 3~). Simultaneously, an enormous proliferation of rough endoplasmic reticu- lum, enlargement and augmentation of mitochondria in addition to hyperplasia indicated high metabolic activity in flounder liver. Shortly after spawning, enlarged autophagic lysosomes and apoptotic bodies containing digested mitochondria and endoplasmic reticulum reflected the degradation of cell com- ponents in adaptation to the new physiological phase of vitellogenic rest (Fig. 3d).

DISCUSSION

The lysosomal stability test, established in mam- malian research by Bitensky et al. (1973) has been successfully applied during the past 15 years by the Plymouth Marine Laboratory (Great Britain) in marine invertebrates. The assessment of lysosomal membrane stability in the digestive gland of marine mussels and snails proved to be a highly sensitive measure for the functional state of the cell (Moore, 1985). Injury of the lysosomal membrane or over- loading of the storage capacity by the toxic com- pounds led to leakage of the hydrolytic lysosomal enzymes into the cytoplasm and nucleoplasm leading to disturbance of cell functions and resulting in degeneration and possibly in neoplasia. No problems were caused by the transfer of the test systems to the fish liver, although, in contrast to the handling of marine mussels and snails, care must be taken during sampling to minimize stress caused by long hawls (exceeding 30min) and oxygen deprivation after catching.

The investigation of seasonal effects on the lyso- somal system clearly demonstrated the participation of the lysosomal system in the synthesis of the yolk-precusor protein vitellogenin in the flounder liver during gonadal growth. Biochemical data on RNA, DNA and glycogen in flounder liver during yolk-precursor protein synthesis (Emmerson and Emmerson, 1976; Petersen and Emmerson, 1977) fit well to morphological alterations observed in this study, described and discussed in detail elsewhere, The lysosomal system in fish liver appears to be more stable against the physiological changes during repro- duction than is documented for that of the marine mussel (Moore et al., 1978). Though the contami- nation effects on the lysosomal system did not seem to be compromised by the demands to the lysosomal system during vitellogenesis, this point should be kept in mind when planning the sampling strategies for biological-effects monitoring.

The results of the test for lysosomal perturbations applied during this study reflected a clear gradient

lctlvlty rmt ac1wty from the Elbe estuary in the direction of the northern 199&!89 1989 Wadden Sea stations. This gradient coincided with

the results of the chemical analyses of heavy metals and organochlorine in flounder liver measured during the present study as indicator substances for the

taminated stations. contamination situation in the Wadden Sea area

Fig. 2. Seasonal variations of lysosomal membrane stability during vitellogenic activity (Oct.-March) and vitellogenic rest (April-Sept.) in flounder liver at two differently con-

126 ANGELA K~HLER

Fig. 3. (a) Liver of flounder from a fess polluted area with small Iysosomes displaying high membrane stability. (b) Dramatically enlarged lysosome in the liver of the Elbe flounder with an already destabihsed membrane. (c) Intralysosomal lipid degradation and transport to the Golgi complexes in phosphohpo- protein synthesis during vitello-genesis, (d) “Giant”-1ysosome and proliferating lysosomes (*) during programmed cell death (apoptosis) after spawning removing now unnecessary cell organelles. Arrow points to just digested ribosomes. LY, lysosome; GC, Golgi complex; M, mitochondria; RER, rough

endoplasmic reticulum; SER. smooth endoplasmic reticulum; N, nucleus (Scale bar = 2.5 pm).

Lysosomal Perturbations in fish liver 127

(U. Harms and K. SiiIIker, unpublished data; Kiihler et al., 1986).

In fish hepatocytes, only few and small lysosomes can be identified under normal conditions. In con- trast, the hepatopancreas of marine mussels and snails possess a well-developed lysosomal system based on the function of endocytosis of nutrients (Owens, 1972). Similar to the present findings in fish liver, a negative relationship between size and number of lysosomes is reported by Moore (1986) in the digestive gland of mussels. Lysosomal enzymes re- leased into the cytosol presumably cause changes in the membrane fluidity resulting in increased fusion rates. Lowe et al. (1981) found that an increase in lysosomal volume accompanied by the formation of pathologically enlarged lysosomes was directly associated with membrane destabilisation in the digestive gland of mussels exposed to oil-derived contaminants.

In contrast to marine invertebrates a clear “two- step response” of the lysosomal system could be detected in flounder liver. The activation of the lysosomal system was reflected by an increase in number and size of lysosomes which accumulated foreign compounds and lipids in the attacked liver. This first step represents an adaptive and protective response to injury.

Decreased membrane stability led to release of degrading enzymes into the cytoplasm and nucleo- plasm, reflecting the overloading or damage of the lysosomal digestive and detoxifying system. This second step of lysosomal response indicated the injury of this central cell compartment, resulting in severe liver lesions. Megalocytic hepatosis and caryomegaly were accompanied by such liver de- generations as necrosis, fibrosis and steatosis and have to be regarded as putative preneoplastic lesions in fish liver (Myers et al., 1987). The significant negative correlation between the lysosomal stability and the extension of liver lesion indicates that the lysosomal stability test clearly reflects the overcharge and breakdown of the detoxifying capacity of liver (Kiihler, 1989b). These results are additionally confirmed by observations during the present study that the preneoplastic changes observed in younger flounder (17-25 cm) progressed towards benign and malignant liver nodules, as cholangioma and cholangiocarcinoma, heiimangioendothelioma and angiosarcoma, such as liver cell adenoma and hepato- cellular carcinoma in female flounder over 30 cm total length.

Based on this results the measurement of lysosomal perturbations in fish liver can be recommended as an integrative biological warning system for the biologi- cal effects monitoring.

Acknowledgements-I want to express my special gratitude to Dr M. N. Moore and Dr D. Lowe (Plymouth Marine Laboratory, Great Britain) for their explanations and train- ing in the use of the lysosomal stability test. The excellent technical assistance by U. Singenstroth, Heike Deisemann, Bjarne Lauritzen and Katja Richter are gratefully acknowl-

edged. This study was financially supported by the Federal Environmental Agency and the provinces of Schleswig- Holstein and Lower Saxony.

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