Nov 14, 1986 - lysosomes, of membrane whorls in the intermembrane space of mitochondria, and .... At arrows, perisinusoidal fat-storing cells; si = sinusoids.
Limnologie ak1ucll · Band/Vol. l · Kinzclbach/Fricdrich (Hg.): Biologie des Rheins Gustav Fischer Verlag · S1u11gan · New York · 1990
Liver pathology in eels (Anguilla anguilla L.) from the Rhine river exposed to the chemical spill at Basle in November 1986 THOMAS B RAUNBECK, P ATRICIA BURKHARDT-H OLM & VOLKER STORCH
Zoologisches Insti tut I, Universität Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg
Keywords Rhine, eel, liver, cytology, histology, chemical spill
Abs tract Morphological changes in the hepatocytes of eels, Anguilla anguilla, foUowing exposure to the chemical spill at Baste in November 1986, were investigated by means of light and electron microscopy. Severe hepatic inj ury is documented in a complexsyndrome comprising pathological alterations in virtually any cell constituent investigated. Glycogen depletion and changes in the amount of lipid inclusions, and, as a consequence, a n::duction in cell size, represent an exhaustion of energy reserves due to acute stress. The invasion of migrating phagocytic cells into hepatic tissue indicates the initiation of a non-specific immune response. A dramalic proliferation of smooth endoplasmic reticulum in combination with a drastic augmentation of lysosomes, autophagic vacuoles, myelinated and lamellar·bodies give morphological evidence of the induction or hepatic detoxification processes, the capacity of which, however, was apparently insufficient. Complete disintegration of the rough endoplasmic reticuJum, atrophy of the Golgi complex, and the emergence of me mbrane-bound crystalline protein inclusions indicate impairment, if not cessation, of efficient hepatic prot ein synthesis and secretion. Further alterations of a possibly specific nature can be demonstrated by tbe occurrence of novel types of lysosomes, of membrane whorls in the intermembrane space of mitochondria, and of crystafüzation processes wi thin the peroxisomal matrix, as weil as by a significant reduction in the number of mitochondrial granu\es. Tbe relative importance and diagnostic value as well as the potential functional implications of the morphological changes observed in the Jiver are discussed.
Kurzfass ung Morphologische Veränderungen io der Leber von Aalen,Anguilla anguilla, infolge des Chemieunfalls bei Basel im November 1986 wurden mit Hilfe liebt- und elektronenmikroskopischer Methoden untersucht. Schwere Leberschädigungen spiegeln sich in einem Syndrom aus Veränderungen in allen untersuchten Ze llbestandteilen: Ein vollständiger Glykogeoabbau, Veränderungen in der Menge der Lipideinschlüsse sowie, als
Folge, eine reduziene Zellgröße sind Ausdruck einer Erschöpfung der Energiereserven der Leber infolge
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akuten Stresses. Eindringende Makrophageo in das Lebergewebe zeigen immunologische Prozesse an. Eioe enorme Proliferation des glatten endoplasmatischen Retikulums in Kombination mit einer starken Zunahme an Lysosomen, Autophagosomen, Myelin- und Lamellarkörpero geben Hinweise auf die Induktion von Abwehrmechanismen und Entgiftungsprozessen in der Leber, deren Kapazität jedoch offensichtlich nicht ausreichend war. Der vollständige Abbau des rauhen endoplasmatischen Retikulums, die Atrophie des Golgi-Apparates sowie das Auftreten membranbegrenzter kristalliner Einschlüsse, vermutlich Proteinkristallen, zeigen tiefgreifende Störungen, wenn nicht den Stillstand von Proteinsynthese und -sekretion in der Leber an. Andere Veränderungen, möglicherweise für einige der bei dem Unfall io den Rhein gelangten Chemikalien spezifisch, ft0den sich io Form von neuen Lysosomentypen, von Membranwirbeln im intermembraoösen Raum der Mitochondrien und Kristallisationsprozcssen in der Matrix der Peroxisomen sowie io der drastischen Abnahme rnitochondrialer Granula. Bedeutung und diagnostischer Wert der Beobachtungen werden ebenso diskutiert wie mögliche Rückschlüsse auf die den morphologischen Veränderungen zugrundeliegenden Mechanismen.
1 Introduction The detrimental effects exerted by the toxic substances released into the Rhine river during the chemical spill at Baste in November 1986 on fish populations were found tobe highly selective. Whereas the strong reaction of species like traut, grayling, perch-pike, pike and others may be explained by increased susceptibility to pesticides, it is difficult to give a reason for the unexpected damage observed in the eel population of the Rhine, since the eel is usually not thought of as a particularly sensitive species. However, detailed toxicological studies using eels as model organisms or even as monitor systems for indication of pollution are scarce (AMIARD-TRIQUET et al. 1987, FERRANDO et al. 1987). A further restraint to attempts to account for the eel kill in the Rhine is that, in fact, not a single or a lirnited number of toxic compounds were spilled, but a complex rnixture of about 30 chernicals including various pesticides, solvents, emulgation aids, stabilizers as weil as primary products (Deutsche Kommission zur Reinhaltung des Rheins, 1986). Residue analysis in killed fish, chemical analysis of the contarninated water and subsequent comparison of toxicological data (LD50 etc.) with analytical data failed to yield conclusive information about the reasons for the fish kill. A completely different approach was used in the present study: Eels, which were immediately exposed to the toxicants, but which had survived the disaster, were collected from the Rhine and analyzed for histologic and cytologic damage by means of light and electron rnicroscopy. As one of the major target organs of pesticides and as the site of various detoxifying enzyrne systems, and due to its proven susceptibility to chemical agents as evidenced by electron microscopy (HINTON et al. 1973, 1978, 1984; HACKING et al. 1978; LIPSKY et al. 1978; MCCAIN et al. 1978; KLAUNTG et al. 1979; SCHOOR & COUCH 1979; HAWKES 1980; ROJIK et al. 1983; BRAUNBECK et al. 1989), the liver was chosen as one model organ. For reviews on toxicological studies in fish liver on the light microscopical
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level see COUCH (1975), WALSH & RIBELIN (1975), SINDERMANN (1980, 1984) and GINGERICH (1982). The present study was designed to compile pathological alterations induced in the eels from the Rhine as a baseline study for subsequent experimental analysis of the syndrome found in the liver and other organs (cf. BURKHARDT-HOLM et al. 1989). The ultimate objective of the project is to make an attempt at elucidating some of the mechanisms active in result of the exposure of the eels, and to evaluate the relative importance of isolated components or simple combinations of the toxicant mixture released into the Rhine in November 1986.
2 Materials and Methods 2.1 Animals
On November 14th, 1986, five adult, but sexually immature eels (Anguilla anguilla L.), which had survived the chemical spill at Basle on November lst, 1986, were collected from a groyne at the right bank of the Rhine river at Ketsch near Heidelberg (Rhine-km 406). Individual length ranged from 35 to 50 cm, mean weight was ca. 100 g; we did not distinguish between male and female specimens. Control animals (median weight about 55 g; mean length approx. 35 cm) were obtained from an eel hatchery in Werl, West Germany, and from the Rhine river upstream of Basle (median weight about 270 g; mean length ca. 55 cm). Control animals were kept in the laboratory without feeding for four weeks in lots of 35 individuals in plastic tanks containing 600 l of permanently aerated tap water (hardness 50 mgfl CaC03 ; ammonia 0.01 mgfl; oxygen saturation between 70 and 90 %; 14±1 °C; pH 7.2 ± 0.2). Water was constantly replaced at a flow rate of 50-60 l/h; no losses occurred during this period. 2.2 Electron microscopy
With reference to possible diurnal variation, all fixation procedures were carried out at midmorning. For electron microscopy, control animals were anesthetized with benzocaine (ethyl-4-amino-benzoate) and perfused in situ via the ventricle, first with physiological saline containing 2 % polyvinylpyrolidone (PVP, Merck, Darmstadt) and 0.5 % procainhydrochloride (Merck) for 2 min to remove blood cells, followed by ice-cold 1.5 % glutaraldehyde and 1.5 % formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M sodium phosphate buffer (pH 7.6) containing 2.5 % PVP for 10-15 min. The posterior portion of the liver was excised immediately after perfusion and immersed as small blocks in perfusion fixative for at least 20 min at 4 °C. The livers of specimens collected from the
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Rhine were fixed by immersion of small liver blocks in ice-cold perfusion fixative for at least 1 h. Subsequently, the tissue blocks of control and exposed specimens were cut into thin slices of 50-80µm using an Oxford vibratome. The fixation was continued in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.6) containing 4 % PVP and 0.05 % calcium chloride at 4 °C for 20 min. After rinsing in cacodylate buffer, the tissue slices were postfixed for 1 hat 4 °C in osm.ium ferrocyanide (l
