Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.     

Enzyme indicator of the immunobiologic activity of antigens

The test-system has been elaborated for the determination of the ability of different antigens in the cultures of the mouse peritoneal macrophages. Measuring an intracellular acid phosphatase activity, reflecting the extent of the cell activation, was taken as a principle of this test-system. With the help of this test-system ability of a few antigens (lipopolysaccharide, polysaccharide, glycoprotein, protein and low molecular weight substances--peptide and glycopeptide) have been studied. It is testified that above mentioned antigens possessed different effects on the acid phosphatase activity.     

Mammalian protein synthesis in the cytoplasm of a plant cell

Using immunochemical and, subsequently, autoradiographic methods, the authors have shown that after the injection of the total fraction of polyribosomes isolated from the rat liver in the Acetabularia cytoplasm a protein interacting with the antiserum against rat thyrosine aminotransferase is synthesized. Prospects of using Acetabularia as a test-system for the translation of heterogenous mRNA and mRNP are discussed.     

Diagnostic test system for detecting the meningococcal antigen by erythro-immunoadsorption

The authors have developed a test-system for detection of group A meningococcal polysaccharide, based on the sandwich erythro-immunoadsorption ultra-microtechnique with the use of the Terasaki plates as a solid-phase carrier. The system, equivalent to ELISA in its high sensitivity and specificity, is more rapid and less expensive, permitting the detection of 1 mg/ml of the antigen in 20 microliter of the tested liquid within an hour. The results of the study of CSF samples from 28 patients with meningococcal infection were in good correlation with the ELISA results. The new test-system is recommended for practical use as a routine technique for the specific diagnosis of meningococcal meningitis and for the control of the effectiveness of the treatment.     

Biosensor analysis of the interaction of potential dimerization inhibitors with HIV-1 protease

Inhibitors of protein-protein interactions are currently considered as perspective prototypes of a new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. HIV-1 protease (HIVp), which is active only as a homodimer form, is the classic example of such enzymes. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on an original biosensor test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by the method of virtual screening, and also some derivatives of glycyrrhizin, triterpenic and steroid glycosides. In the results of this study we have found one compound, which preferentially interacts with HIVp monomers and inhibits in vitro activity of this enzyme with the IC₅₀ value of about 10^?6 M.     

Using comet assay for the evaluation of biological activity of heavy metal salts in eucaryotic cells

The test-system for the examination of different factors modifying effect on heavy metals biological activity was chosen. Nickel-induced DNA damage was examined in the chosen test-system.     

An immunochemical analysis of the induction of cytochrome P-450 isoforms in the liver of fresh-water fishes by 3-methylcholanthrene, beta-naphthoflavone and aroclor 1254

The cytochrome P-450 isoforms have been studied in liver microsomes of some fish species from Lake Baikal. Using the inhibitory analysis of microsomal monooxygenase activities carried out by the specific polyclonal antibodies it has been shown that 3-methylcholanthrene, beta-naphthoflavone and arochlor 1254 induce isoforms immunologically related to cytochrome P-488c but not to the rat cytochrome P-450b in fish liver microsomes. The immunologic identity in isoforms of fish and rat cytochromes induced by methylcholanthrene has not been revealed. A possibility to use the method of the inhibitory analysis of fish microsomal activities by specific antibodies to the rat cytochrome P-450 isoforms for biomonitoring and biotesting of polycyclic hydrocarbons and polychlorinated biphenyls in aquatic systems is discussed.     

Heavy Metals in the Tissues of Commercially Important Fish of Amurskii Bay,Sea of Japan

The concentrations of iron, copper, zinc, manganese, cadmium, and mercury in tissues and organs of Pacific herring, Far Eastern navaga, and spotted flounder from Amurskii Bay, Sea of Japan, were determined using an atom-absorption method. The distribution of these elements has been studied in organisms of the fish. The greatest concentration of iron, copper, cadmium, and mercury is found in the liver of the fish, manganese is mostly accumulated in the bone tissue, and zinc is found in the skin. Some specific features of metal accumulation in the fish of Amurskii Bay have been revealed. For example, the concentration of iron in the liver of herring and flounder significantly increased the mean concentration known from other areas. A sanitary–hygienic evaluation is provided for the recent levels of metal concentrations in these three species of commercially important fish.     

Application of the SOS umu-test in detection of pollution using fish liver S9 fraction

1. The possibility of Aroclor 1254 and beta-naphthoflavone treated fish Mugil auratus and fish sampled in low and high polluted areas to convert some premutagens to active intermediers in the SOS umu-test have been investigated. 2. Genotoxicity of Aflatoxin b1 differed markedly upon activation with liver S9 fractions from fish with different pollution histories, with the highest activation potency in fish living near a fish cannery. 3. Inhibition of umu gene expression by 7,8-benzoflavone in vitro clearly demonstrates a cytochrome P-450 mediated activation of aflatoxin b1. 4. 2-Aminoanthracene and 2-aminofluorene were weakly activated to genotoxic products and the induction of umu gene expression could be detected only in the presence of S9 fractions from fish treated with beta-naphthoflavone and Aroclor 1254 in the laboratory. 5. The capability of S9 from fish living near a fish cannery to convert 2-aminoanthracene and 2-aminofluorene was not observed.     

Channa punctata brain metallothionein is a potent scavenger of superoxide radicals and prevents hydroxyl radical-induced in vitro DNA damage

Mammalian brain metallothioneins (MTs) have been shown to scavenge free radicals. However, a similar role for fish brain MT has not been established yet. Previously, we have reported that MT from the liver of a freshwater fish, Channa punctata Bloch, had free-radical-scavenging activity in vitro. In this study, we report on the induction of MT in brain and other tissues of C. punctata treated with a low concentration of zinc chloride. We partially purified MT (Zn-MT)-rich fraction from the brain and studied its free-radical-scavenging and DNA damage attenuating effects. Zinc exposure showed significant MT induction in brain, gill, kidney, and liver. C. punctata brain MT efficiently scavenged superoxide radicals and also attenuated hydroxyl radical-mediated DNA damage. These findings suggest that fish brain MT has a free-radical-scavenging activity, and its expression may be regulated in response to stress and chemical exposure. C. punctata has been identified as a potent biomarker fish species. It is suggested that this fish species may be a good model for the study of MTs with regard to their regulatory and biomarker functions.     

The use of the polyene-resistant mutation model in Saccharomyces as a test-system for detecting genetically active substances

The simple test-system has been developed to register mutagenic activity of different chemicals using a model of forward polyene-resistant mutations in Saccharomyces cerevisiae.     

A study of the swimming performance of the Crucian carp Carassius carassius (L.) in relation to the effects of exercise and recovery on biochemical changes in the myotomal muscles and liver

A study has been made of the maximum sustained swimming speed of Crucian carp Carassius carassius (L.) using a fixed velocity technique. The data obtained from swimming tests on 214 carp have been analysed using the method of probit analysis. The 50% fatigue level for 13–16 cm fish acclimated to 9.5±0.6°C has been estimated to be 3.35 lengths/sec. Biochemical measurements have been made on the red and white myotomal muscles and liver of fish subjected to both varying intensities of sustained swimming and short periods of vigorous swimming. Free creatine was found to increase only during high speed swimming in the white muscle. Elevated lactate concentrations occurred at both low and high sustained swimming speeds in the red superficial muscle but not during short periods of strenuous exercise. Glycogen depletion from the red musculature also only took place at the sustained swimming speeds investigated. The reverse situation was operative in the white muscle, significant glycogen depletion occurring only at the highest swimming speed studied. Lactate levels were only significantly different from non-exercised fish in the fish swimming at the higher velocities. The effects of periods of recovery following 200 min of sustained swimming were also investigated. White muscle lactate was at a higher level than non-exercise fish 5 h post-exercise, while both red muscle glycogen and lactate rapidly returned to pre-exercise concentrations. Biochemical measurements on the myotomal muscle types have been discussed in relation to the swimming performance of the fish and the division of labour between red and white fibres.     

The evaluation of the genotoxic effects of samples of industrial and domestic sewage. II. The mutagenic and recombinant effects of sewage in the soybean (Glycine max. (L.)) test system

The genotoxicity of the industrial and domestic sewage has been studied on the soybean test-system Glycine max (L.). It makes it possible to take into account different types of genetic disturbances according to appearance of various spot types on sprout leaves (somatic mosaicism).     

Prevalence of the gene encoding the outer membrane lipoprotein LipL32 in leptospires of different taxons

Primers flanking the fragment sized 677 bp have been constructed on the basis of nucleotide sequences of the gene encoding the outer membrane lipoprotein LipL32. PCR-analysis was used to reveal the prevalence of gene lipL32 among 73 Leptospiraceae family strains representing different genera and genomic species. The gene lipL32 appeared to be conservative across the pathogenic species. In contrast, it was not detected in the genome of nonpathogenic free-living leptospires. Thus the developed PCR test-system with primers LEP21/LEP22 may be efficiently used to differentiate these two distinct ecological groups of leptospires.     

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.     

Dietary Fish Oil Inhibits Pro-Inflammatory and ER Stress Signalling Pathways in the Liver of Sows during Lactation

Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.     

Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.     

Biological properties of an asparaginase-glutaminase preparation from Pseudomonas fluorescens in cell cultures]

Specific L-asparaginase activity and non-specific cytotoxicity of asparaginase-glutaminase preparation from Pseudomonas fluorescens were studied. Two cell lines, i.e. the asparaginase-dependent (Berkitt lymphoma cells) and the asparaginase-independent (the ovary cancer cells) were used as the test-system. Incorporation of 3H-timidine into DNA was used as the criterion of the drug effect on the cells. Krasnitin was used as the reference preparation. The preparation of asparaginase-glutaminase was inferior to krasnitine by its specific antitumour asparaginase activity and superior to it by the general cytotoxicity in the cells of CaOv. With the help of the above test-system it is possible to study the specific asparaginase activity of the drugs containing L-asparaginase. For studying the specific glutaminase properties it is necessary to develop another cell test-system.     

Early diagnostics of hemorrhagic fever with renal syndrome on the basis of the use of Pumala virus recombinant nucleocapsid protein

Pumala virus recombinant nucleocapsid protein was used for the early diagnosis of haemorrhagic fever with renal syndrome. Specific IgM in the sera of patients could be determined by the IEA technique as early as on days 2-3 from the onset of the disease. The diagnostic effectiveness of the test-system was 95% and its specificity was 98%.