Rolf A. Andersen

Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode

Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (Salmo trutta) during a run-off episode in two rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 µg/l at the low water level to 41 µg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90-170 ng/l and those for Zn were 49-91 µg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss).

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZs mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 microg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.

Brown trout (Salmo trutta) metallothioneins as biomarkers for metal exposure in two Norwegian rivers

The potential use of the metal binding protein metallothionein (MT) as a biomarker for trace metal exposure has been evaluated in brown trout (Salmo trutta)The potential use of the metal binding protein metallothionein (MT) as a biomarker for trace metal exposure has been evaluated in brown trout (Salmo trutta)The potential use of the metal binding protein metallothionein (MT) as a biomarker for trace metal exposure has been evaluated in brown trout (Salmo trutta).

Gill metal binding and stress gene transcription in brown trout (Salmo trutta) exposed to metal environments: The effect of pre‐exposure in natural populations

Brown trout (Salmo trutta) from two native populations from the Røros area in Central Norway, acclimated in mining-affected habitats to different levels of Cd/Zn and Cu, together with trout from a nearby unaffected river (reference) were transferred to a nearby lake with higher levels of Cu, Cd, and Zn than those in their respective native rivers. This experiment was conducted to gain information about the underlying resistance mechanisms developed in fish exposed to metal environments. The focus was on gill metal accumulation and transcription of the metal-responsive stress genes metallothionein-A (MT-A), Cu/Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and heat shock protein 70 (HSP-70). The only shared response shown between the three groups after transfer were Cu accumulation and MT-A induction. The Cu-acclimated trout produced mucus to reduce the uptake of Cu into the gills. The MT-A levels were highest in the Cd/Zn-acclimated trout both before and after transfer. Before transfer, antioxidant transcription (SOD and GPx) was higher in gills of Cu-acclimated compared to the Cd/Zn-acclimated trout, but increased transcription of antioxidant stress genes was observed after transfer in both metal-acclimated groups. The metal-acclimated trout groups also showed an increase in the transcription of HSP-70. Compared to the reference population not previously exposed to metals, stress gene transcription increased faster in the metal-acclimated populations. The exception was induction of CAT, which appeared to be depressed after transfer in Cd/Zn-acclimated trout. The data indicate that acclimation to chronic metal exposure involves different strategies to cope with different metals and that these strategies involve both physiological mechanisms (mucus production) as well as metal-related stress gene transcription.

Mushrooms from two metal-contaminated areas in Norway: occurrence of metals and metallothionein-like proteins

This study tested the hypothesis that concentrations of metallothionein-like (MT-like) proteins in fruiting bodies of wild-growing macrofungi reflect exposure to the metals Cd, Cu and Zn. Screening for these proteins was done by taking 44 samples of seven species of macrofungi from two metal-polluted areas in Norway, namely Odda and Sulitjelma. Concentrations of Cd, Cu and Zn were measured in fruiting bodies and topsoil, the highest of which were observed in fruiting bodies – 126 μg g−1 for Cd, 427 μg g−1 for Cu and 396 μg g−1 for Zn. A large intra- and inter-specific heterogeneity in metal uptake was displayed between fruiting bodies from the same soils. Cd,Zn–MT-like proteins were detected in all samples, except one stipe of Boletus edulis, whereas only 72% of the samples contained detectable levels of Cu–MT-like proteins. However, concentrations of MT-like proteins did not reflect variations in metal concentrations. Low content of cysteine in Cd-binding protein fractions from Amanita muscaria collected in Odda indicates that no proteins of the metallothionein family were present in this species. Onthe contrary, these protein fractions share several characteristics with cadmium–mycophosphatins.

Heavy metal content in liver and kidneys of grey seals (Halichoerus grypus) in various life stages correlated with metallothionein levels: Some metal—binding characteristics of this protein

Liver and kidneys from grey seals (Halichoerus grypus) in their natural habitats in Norwegian waters contain high levels of Cd and Hg as well as metallothionein (MT). The data show significant placental transfer of heavy metals in seals. The MT concentrations were higher in fetus liver and kidneys than in the liver and kidneys of juveniles and adults. A significantly higher proportion of the total Cd and Cu, as compared to Zn and Hg, in fetal liver was associated with MT. Only low amounts of metals and MT were found in juveniles. In kidneys, little metal was associated with MT, although high amounts of Cd were bound in fetal stages. Positive correlations were found between total metal, MT content, and MT-bound metals, particularly for Cd and Zn. Grey seal MT may have higher affinity to Zn than to Cd. The use of seal MT as a biomarker for environmental heavy metal exposure seems promising.

Preparation of metallothionein from rat liver and studies of its properties with respect to use as a standard in gel permeation chromatography, polyacrylamide gel systems, autoradiography and Western blotting.

1. A simple method for preparation of metallothionein (Mt) I and II has been developed for the purpose of making standards for use in various biochemical systems and in antibody production. 2. The theoretical content of SH groups in a Mt protein; assuming the mol. wt to be 10,000 and each molecule to contain 20 SH groups was found to be 7.1 and 7.7 times higher than for our purified Mt I and II, respectively. 3. In our native polyacrylamide gel system Mt I ran ahead of Mt II, while the two Mt forms were not separated in the Laemmli SDS system in which it behaved as a protein with mol. wt 10,000. In both gel systems, however, Mt I stained as a very faint band in comparison to Mt II, despite equal absorbance at 254 nm and Cd binding capacity. 4. Compared to staining of polyacrylamide gels with Coomassie Brilliant Blue less than 1/50 parts (1 ng) of the protein could be easily seen after silver staining. 5. It was found that Mt may undergo spontaneous modification, polymerization and loss of metal binding properties. 6. Spontaneous modification and polymerization reduced the antigenic properties of our purified Mt. Only Mt II appeared to be immunologically active.

Effects of a homoanatoxin-a-containing extract from Oscillatoria formosa (Cyanophyceae/cyanobacteria) on neuromuscular transmission

Experimental investigations were carried out with cultured and lyophilized material of the toxigenic strain Oscillatoria NIVA-CYA 92. This organism is classified as Phormidium formosum (Boryex Gom.) Anagnet kom. Aqueous extracts of the algal material, containing the bioactive secondary amine alkaloid 2-(propan-1-oxo-1-yl)-9-azabicyclo(4,2,1)non-2ene (homoanatoxin-a) in an amount of 2.57 micrograms/mg lyophilized material, were tested for acute in vivo toxicity in mice, and for toxicity on neuromuscular transmission by means of electrophysiological methods on the isolated phrenic-nerve hemidiaphragm from rat and in the frog rectus abdominis assay. Acute toxic effects in mice were observed by i.p. and oral (by gavage) administration. Lethal doses were in the range 112-225 and 1125-2250 mg of freeze-dried algal material per kg body weight (i.e. 288-578 and 2890-5780 micrograms homoanatoxin-a/ kg body weight), respectively. The nerve-initiated muscle contractions in the rat diaphragm were blocked by about 0.125 mg cyanophyte material per ml bath solution (i.e. 0.32 microgram homoanatoxin-a/ml or 1.8 microM), but muscle contractions, although slightly reduced, could still be elicited by direct electrical stimulation of the muscle. The compound action potentials recorded from the main phrenic-nerve trunk were not affected. An additive blocking effect on partly curarized preparations was observed and cholinesterase inhibition by physostigmine (eserine) transiently augmented the muscle twitch contraction in preparations partly blocked by the extract. Intracellular recordings from single muscle fibers of homoanatoxin-a-treated rat hemidiaphragm disclosed a partial depolarization and a decrease in the endplate potential to subthreshold level simultaneously with a decrease and then complete disappearance of the miniature endplate potentials. The neuromuscular transmission block was reversed by washing. The extract produced muscle contractions in the frog rectus abdominis assay. Homoanatoxin-a in the algal material was readily absorbed from the gastrointestinal tract in mice. Blockade of the neuromuscular transmission of the respiratory muscle may partly explain the acute toxic effects observed in mice. Thus, the main target of the homoanatoxin-a action at the mammalian neuromuscular junction was traced to the postsynaptic nicotinic acetylcholine receptor channel complex, where it reduced the sensitivity to the transmitter substance.

Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type

Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.