H.J. Herwig

Time-course and dose-response of the apparent induction of the cytochrome P450 monooxygenase system of pyloric caeca musomes of the female sea star Asterias rubens L. by benzo[a]pyrene and polychlorinated biphenyls

Abstract A study was made on the time-course of putative monooxygenase (MO) induction and the dose-response relationships for the effects of 3,3′,4,4′,5-pentachlorobiphenyl (IUPAC congener 126), 2,3′,4,4′,5-pentachlorobiphenyl (congener 118), 2,2′,4,4′,5,5′-hexachlorobiphenyl (congener 153) and benzo[a]pyrene (BaP) on MO-system activities in female sea stars Asterias rubens L. Sea stars received a single injection of PCB or BaP at different concentrations. Cytochrome P450 concentration and activities of NADPH-cytochrome c (P450) reductase, BaP hydroxylase (BPH) and prenenolone hydroxylase were determined in musomal fractions of the pyloric caeca. Apparent induction of BPH activity by PCB was observed only with CB-126 (3-methylcholanthrene (3-MC)-type inducer) at 10 μmol/kg. BPH activity was not affected by CB-153 (phenobarbital-type inducer) or CB-118 (mixed-type inducer). BaP (3-MC-type inducer) elicited a clear dose-dependent BPH induction in the range 10–160 μmol/kg. The maximum observed increase in BPH activity was about 350%. In time-course studies with CB-126 and BaP, highest BPH activities were found 3–4 days after injection. HPLC analysis of musomal BaP metabolites revealed that the elevated metabolism was shifted towards phenol production. PCB or BaP had no effect on the level of cytochrome P450 or the NADPH-cytochrome c reductase activity. All three PCBs and BaP inhibited pregnenolone hydroxylase activity, but no dose-response relationships were found. Overall the results suggest that the echinoderm MO system is inducible in response to exposure to cytochrome P450 1A-type inducers. However, the mechanism of induction remains unclear.

Cytochrome P-450 monooxygenase system and benzo(a)pyrene metabolism in echinoderms

A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb5 and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 ×g) fractions, but MFO system components were also found in the mitochondrial (12 000 ×g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.

Effects of cadmium and polychlorinated biphenyls (Clophen A50) on steroid metabolism and cytochrome P-450 monooxygenase system in the sea star Asterias rubens L.

Abstract A study was made on the effects of long-term cadmium or PCB exposure on the steroid metabolism and the cytochrome P-450 monooxygenase system of the sea star Asterias rubens. Sea stars were exposed to 25 μg/l cadmium or fed with PCB (Clophen A50)-exposed mussels containing 26 μg σ8-PCBs/g lipid, which was six times higher than the PCB levels in control mussels. Twelve weeks of exposure resulted in cadmium and PCB levels in the pyloric caeca which were comparable to those found in sea stars collected from the Western Scheldt (SW Netherlands) or in animals that had been fed with mussels from this heavily polluted estuary. Cadmium and PCB exposure caused significant reductions in the levels of progesterone and testosterone in the pyloric caeca of male and female sea stars. Steroid levels in the gonads had not changed after cadmium exposure, whereas after twelve weeks of PCB exposure elevated levels of testosterone were found in testes and ovaries. Both cadmium and PCBs caused a decrease in the content of cytochrome P-450 and cytochrome b5 in pyloric caeca musomes. Benzo[a]pyrene hydroxylase activity was elevated by PCB exposure but had not changed after exposure to cadmium. Microsomal NADPH-cytochrome c reductase and steroid conversion activities were not significantly influenced by cadmium or PCB exposure, although a slight reduction of steroid metabolism was observed.

Bioaccumulation and histochemical localization of cadmium in Dreissena polymorpha exposed to cadmium chloride

Dreissena polymorpha, a freshwater bivalve, was exposed to dissolved CdCl2 (100 μg Cd/l) for periods of 1, 2, 3 or 4 wk. Cadmium accumulation was followed by atomic absorption spectrophotometry and the localization of Cd was investigated with a sulphide-silver technique (SST). During the exposure period the soft body accumulated Cd in a linear way, while the shells reached a saturation level after 3 wk. In exposed mussels treated with the SST, reaction products were present in nearly all tissues. The cellular localization and the amount of reaction products varied according to the type of tissue and the duration of exposure. After one week, reaction products indicative for the presence of free or loosely bound Cd were present in granular structures and in the cytoplasm of all epithelia that had been in direct contact with the Cd-contaminated water. After longer exposure times, the epithelial cells as well as the underlying connective tissue became completely filled with reaction products. In the digestive tract, reaction products were confined mainly to granular structures in the epithelial cells. They became prominent after 3 or 4 wk of exposure. Among the internal organs, the excretory system showed a reaction pattern largely comparable to that of the epithelia which had been in direct contact with the Cd-contaminated water. Apart from the pericardial gland, which represents the site of ultrafiltration, the appearance of reaction products in the cytoplasm of the kidney cells was retarded. In the epithelial lining of the gonads and in neural tissue, diffusely distributed reaction products were observed only after longer exposure times. Gametes and muscular tissue, except for the ventricular muscle, remained free of reaction products. Structural damage related to the Cd-exposure was restricted to the pericardial gland.

Effects of cadmium in freshwater clams. III. Interaction with energy metabolism inAnodonta cygnea

Freshwater clams,Anodonta cygnea, were exposed to cadmium, at 50 ppb (μg/L), to investigate the effects of semi-chronic exposure on energy metabolism. Parameters examined included: adenylate energy charge (AEC), glycogen content, blood glucose and protein concentration, the accumulation of anaerobic metabolic end products,viz. lactate and succinate, and mitochondrial NADH-oxidase activity. In all tissues, AEC was significantly lowered after 12 weeks. Glycogen contents of the separate organs, except for the gills, were diminished to one half those of control animals. Hemolymph glucose increased between 4 and 8 weeks, whereas protein in hemolymph steadily decreased, to about one half the concentration of controls at 12 weeks. Lactate increased in mantle and midgut gland, whereas in gill only an initial accumulation was found after 2 weeks of exposure. Succinate concentrations increased in all organs between 4 and 8 weeks of Cd exposure. Thereafter, no further accumulation occurred. From the onset of exposure to cadmium there was a gradual reduction of NADH-oxidase activity of gill mitochondria, down to 60% of the control value after 12 weeks. The data indicate an impairment of oxidative carbohydrate metabolism that is brought about by a metabolic blockade, rather than by (partial) anaerobiosis as a consequence of shell closure.

Cadmium accumulation and metallothionein-like proteins in the sea star Asterias rubens

Sea stars were exposed for four months to cadmium at 50 μg Cd/L. The cadmium content of stomachs, pyloric caeca, gonads and body wall was determined after 2, 4, 6, 8, and 16 weeks of exposure. The highest accumulation rates were found for body wall and pyloric caeca. The lowest accumulation rate was found for the gonads. In all tissues cadmium accumulation showed saturation kinetics. The body wall contained the largest part of the accumulated cadmium.Sephadex G-75 gel filtration experiments demonstrated the presence of metal-binding, metallothionein (MT)-like proteins with a molecular weight of 10.8 kD in cytosolic fractions of pyloric caeca from unexposed sea stars. The MT-like proteins contained mainly zinc and a small amount of copper. During exposure, the MT-like proteins accumulated cadmium at a relatively high rate, but a steady state was reached after 8 weeks, indicating thatin vivo, the Cd-binding capacity of these proteins was limited. When pyloric caeca cytosol from unexposed sea stars was incubated with cadmiumin vitro, the MT-like proteins could bind larger amounts of cadmium thanin vivo during the Cd exposure. After 16 weeks of Cd exposure, thein vitro Cd-binding capacity had hardly increased.Low-molecular-weight metal-binding proteins were absent in cytosolic fractions of the testes, ovaries and oocytes from Cd-exposed sea stars, even after four months of exposure. The cadmium accumulated in the gonads was bound exclusively to high-molecular-weight proteins, which accumulated cadmium at a relatively low rate, compared to the MT-like proteins in the pyloric caeca. The absence of MT-like proteins in the gonads may explain the low cadmium accumulation rates found for these organs, but as a consequence, the accumulated cadmium is not detoxified.

Cytopathological investigations of digestive tract and storage cells in Daphnia magna exposed to cadmium and tributyltin.

Histopathological effects on the ultrastructural organization of digestive tract and storage cells of Daphnia magna were examined after a 7-day exposure to cadmium or tributyltin chloride. No distinct cytological changes were noticed in the above-mentioned tissues of D. magna exposed to 10 μg Cd/l. In animals exposed to 20 μg Cd/l, however, there was a slight decline in the glycogen content of the storage cells and some structural changes in the mitochondria of these cells. By contrast, in daphnids exposed to 1 or 5 μg TBTC/l the ultrastructural integrity of storage cells appeared to be seriously affected. In addition, conspicuous lipid accumulations were observed in posterior gut epithelium and in a specific haemolymphatic cell-type of TBTC-exposed D. magna. The results of this histopathological investigation are discussed within the framework of our toxicity studies on the effects of cadmium exposure in Daphnia.

Interference of polychlorinated biphenyls (Clophen A50) with gametogenesis in the sea star, Asterias rubens L.

Abstract A study was made on the accumulation, distribution and effects of polychlorinated biphenyls (PCBs) in the sea star Asterias rubens . Sea stars were exposed for five or seven months to PCBs by a diet of mussels which had been fed with Clophen A50-contaminated algae. Seasonal changes of the PCB concentrations in pyloric caeca from unexposed sea stars and differences between the PCB contents of different organs were found to be related mainly to variations and differences in the lipid contents. During PCB exposure via the foodchain, an almost linear increase of the PCB concentration in pyloric caeca and gonads of the sea star was found. After seven months of exposure, the gonads of male and female sea stars contained respectively 26 and 43% of the total amount of accumulated PCBs. The PCBs were present in the oocytes and spermatozoa from exposed animals at levels comparable to those found for whole gonads. The PCB levels in experimentally exposed sea stars were found to be similar to those in animals collected directly from the polluted Dutch Western Scheldt. In PCB-exposed female sea stars, the gonad indices at the end of the reproductive cycle were significantly lower than in unexposed animals. No effects were found on the size of the oocytes or on the biochemical composition with respect to the overall glycogen, lipid or protein levels and SDS-PAGE protein patterns. The gonad indices of PCB-exposed male sea stars were not different from those of unexposed animals.

Effects of cadmium on metal composition and adenylate energy charge in the sea star Asterias rubens L.

Sea stars,Asterias rubens, were exposed to 200 μg Cd/L or fed with mussels which contained about 70 μg Cd/g dry wt. After 5 weeks, cadmium in the pyloric caeca of directly and indirectly exposed sea stars had reached levels of 12 and 9 μg Cd/g dry wt, respectively. For both types of exposure, a reduction of 30% of the zinc levels in the pyloric caeca was found, which was correlated with a comparable displacement of zinc from the metallothionein-like proteins. Copper levels were increased in the pyloric caeca of directly exposed sea stars.In gonads, stomachs, and body wall of directly exposed sea stars, cadmium concentrations were 4 to 9 times higher than those in animals fed with Cd-contaminated mussels. Cadmium exposure also affected metal composition in these tissues. The ovaries contained relatively large amounts of zinc. Gel filtration chromatography revealed that this zinc and the accumulated cadmium were distributed over a large range of high-molecular-weight proteins.Both direct and indirect cadmium exposure resulted in a small, but significant decrease of the adenylate energy charge (AEC) in the pyloric caeca. In the gonads, no effect of the cadmium exposure could be demonstrated on the AEC, but in the ovaries a reduction of the adenylate pool was found.In semi-field experiments, stars were exposed to 25 μg Cd/L or fed with mussels collected from the heavily polluted Dutch Western Scheldt. After 6 months of direct or indirect exposure, cadmium in the pyloric caeca had reached comparable levels of 8 and 7 μg/g dry wt, respectively. In the gonads of sea stars that had been fed with Western Scheldt mussels, cadmium levels were lower than those in directly exposed sea stars, but still about a factor 10 higher than those in unexposed animals. Exposure of sea stars to 25 μg Cd/L for three months, had no effects on the levels of zinc, copper and magnesium or on the AEC in the pyloric caeca.

Effects of cadmium in freshwater clams. II. Ultrastructural changes in the renal system ofAnodonta cygnea

An ultrastructural study was made of the renal system of freshwater clams,Anodonta cygnea, that had been exposed to cadmium chloride (50 μg Cd/L) for 12 weeks. By stereological analysis an extended lysosomal system and a decreased number of mitochondria was apparent in the epithelial cells lining the proximal compartment of the kidney. The increase of the lysosomal system was mainly accountable to the appearance of a distinct type of lysosome, that accumulated in the apical cell region. The decrease of the mitochondrial population was accompanied by a considerable swelling of the individual mitochondria. Finally, a severe reduction of the glycogen stores was noticed. Similar, but less obvious, changes occurred in the distal kidney compartment. The results suggest that long-term exposure ofAnodonta cygnea to cadmium stimulates the lysosomal system and disturbs the function of organelles involved in the energy metabolism of resorptive kidney cells.