J. Nemcsók

Biochemical and morphological changes in carp (Cyprinus carpio L.) liver following exposure to copper sulfate and tannic acid

As a consequence of human activity various toxicants reach the aquatic ecosystems; humics may interact with them and may change their toxicity. Many fish are exposed to a considerable concentration of humics and pollutants. Because of paucity of data on the biochemical action of tannins in the presence of the fungicide CuSO4 a comparative study was undertaken. The alterations of redox-parameters in carp liver were monitored and tissue necrosis was followed by measuring the plasma transaminase activities and by electron microscopy. Tannic acid, a representative phenolic/humic compound, exerted prooxidant effects in carp, which may be partially due to formation of prooxidant intermediates/end-products via its biotransformation. Alternatively, tannic acid may partially inhibit the antioxidant enzymes of fish. The response to CuSO4 was more severe. Although tannic acid alone acted as a prooxidant in fish, electron micrographs demonstrated that it reduced the necrotizing effect of copper, which may be due to the complexing activity of tannic acid with the biomolecules of the hepatocytes and to the H2O2-degrading activity of tannin-CuSO4 combination. Our results indicate that the heavy metal-detoxifying capacity of tannin may be significant; however, tannin-exposure alone or combined with metals may be toxic for fish due to enzyme inhibition and oxidative stress induction.

Identification of two hsp90 genes in carp.

Two hsp90 cDNA isoforms (hsp90alpha and hsp90beta) were isolated from the common carp (Cyprinus carpio). Gene-specific probes and primers were selected and used in Northern blot hybridization and RT-PCR reactions to measure the basal hsp90 mRNA levels and to follow the inducer-specific expression of the hsp90 genes in different tissues during in vivo studies. The hsp90beta gene is largely constitutively expressed at a fairly high level in all the examined tissues (brain, liver and kidney) and is slightly inducible by an elevated temperature. Hsp90alpha mRNA is present in the brain, but is hardly detectable in the kidney and liver of unstressed animals. In the brain, this gene is greatly upregulated following thermal stress, whereas in the liver and kidney heat shock has only minor effects on its expression. Hsp90alpha, but not hsp90beta, responds to an elevated level of Cd in a dose-, time- and tissue-dependent manner.

Expressions of heat shock and metallothionein genes in the heart of common carp (Cyprinus carpio): Effects of temperature shock and heavy metal exposure

Heat shock proteins (HSPs) and metallothioneins (MTs) play important roles in protection against environmental stressors. The present study analyzes and compares the regulation of heat shock ( hsp70, hsc70-1 and hsp90alpha ) and metallothionein (MT-1 and MT-2) genes in the heart of common carp, in response to elevated temperature, cold shock and exposure to several heavy metal ions (As 3+ , Cd 2+ and Cu 2+ ), in whole-animal experiments. Among these metals, arsenate proved to be the most potent inducer of the examined stress genes; the hsp90alpha and MT-1 mRNA levels were elevated 11- and 10-fold, respectively, after a 24-h exposure. In contrast, Cd 2+ at 10 mg/L had no impact on the expression of hsp90alpha , and the MT genes also proved to be rather insensitive to Cd 2+ treatment in the heart: only a 2-2.5-fold induction was observed in response to 10 mg/L Cd 2+ . Heat shock resulted in a transient induction of hsp70 (19-fold) and hsp90alpha (15-fold), while elevated temperature had no effect on the expression of the MTs. Direct cold shock induced hsp70 expression (14-fold), while the hsp90alpha (26-fold) and MT-2 (2-fold) expressions peaked after the recovery period following a direct cold shock. The five stress genes examined in this study exhibited a unique, tissue-specific basal expression pattern and a characteristic sensitivity to metal treatments and temperature shocks.

Infraterminal spreading and extrajunctional expression of nicotinic acetylcholine receptors in denervated rat skeletal muscle

Abstract Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds.

EXPRESSION OF TWO METALLOTHIONEIN GENES IN DIFFERENT BRAIN REGIONS OF COMMON CARP

The expression pattern of two metallothionein (MT) genes in response to temperature shock and exposure to Cd(2+) was investigated in the brain of common carp ( Cyprinus carpio ), in whole-animal experiments. The changes in the levels of MT-1 and MT-2 mRNA in the olfactory lobe, midbrain and cerebellum were followed by semiquantitative RT-PCR. The inducibility of the two MT genes was brain region and stressor-specific. Cd(2+) affected mostly the expression of MT-2, while the level of the MT-1 transcript did not change significantly in any of the brain regions examined. Moreover, the MT-2 expression was regulated spatially; MT-2 was induced significantly more strongly in the olfactory lobe than in the cerebellum or midbrain. A sudden temperature drop mainly affected the expression of the MT-1 gene; after 5 h of cold shock, the MT-1 mRNA level was about 25% of the basal value in the cerebellum and the midbrain region. The MT-2 expression did not change significantly during this treatment.

Parabrachial origin of calcitonin gene-related peptide-immunoreactive axons innervating Meynert's basal nucleus

Meynerts basal nucleus is innervated by calcitonin gene-related peptide (CGRP)-immunoreactive axons synapsing with cholinergic principal cells. Origin of CGRP-immunopositive axons was studied in the albino rat. Since beaded axons containing the nicotinic acetylcholine receptor (nAChR) are also present in the basal nucleus, the microstructural arrangement raises the question whether or not an interaction between CGRP and nAChR exists like in the neuromuscular junction. We found that electrolytic lesion of the parabrachial nucleus results in degeneration of CGRP-immunoreactive axons in the ipsilateral nucleus basalis and induces shrinkage of principal cholinergic neurons while the contralateral nucleus basalis remains intact. Electrolytic lesions in the thalamus, caudate-putamen, and hippocampus did not induce alterations in Meynerts basal nucleus. Disappearance of CGRP after lesions of the parabrachial nucleus does not impair presynaptic nAChR in the basal nucleus, suggesting that, unlike in the neuromuscular junction, CGRP is not involved in the maintenance of nAChR in the basal forebrain. It is concluded that the parabrachial nucleus is involved in the activation of the nucleus basalis-prefrontal cortex system, essential in gnostic and mnemonic functions.

Effects of crude oil and oil fractions on the liver P450-dependent monooxygenase activities and antioxidant defence system of different freshwater fish species.

The effects of crude oil (Szeged-Algyo, Hungary) and oil fractions (F1: rich in aromatics; F2 fraction: free from aromatics) were investigated on liver CYP1A isoenzymes and antioxidant defence system following their i.p. injection into different freshwater fish species: carp (Cyprinus carpio L.), silver carp (Hyphothalmichtys molitrix V.), and European eel (Anquilla anquilla). A dose of 2 mL kg -1 crude oil enhanced EROD activity 8-fold in carp and only 5-fold in eel after 3 days. Oil fraction F1 caused only a 2-fold induction in EROD activity only in carp, while F2 fraction caused significant inhibition in all three investigated fish species. The antioxidant parameters [lipid peroxidation (LP), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH)] were measured following the treatment. A decrease of 50% in CAT activity was observed after oil treatment. The GSH level enhanced, resulting the protective effects against LP. The same dose of crude oil but a longer duration time resulted in lower CYP1A induction in carp and antioxidant parameters had returned close to control. In all treatments the EROD isoenzymes proved to be more sensitive and the effects of oil treatment showed species to be different. Carp proved to be more sensitive than eel or silver carp.