Comparative antioxidant enzyme study in freshwater fishes. II. Distribution of antioxidant enzymes and lipid peroxidation in omnivorous fish organs

Comparative studies were performed on the activities of superoxide dismutases, catalase and glutathione peroxidase in organ homogenates from three omnivorous fishes, the barbel, crucian carp and common carp. The lipid peroxidation and protein contents of organ homogenates were also compared. These comparative measurements primarily provide control values for subsequent toxicological examinations. The highest total superoxide dismutase activities were found in the liver or roe, kidney, heart and spleen in every cases. The antioxidant enzyme activities and other studied parameters of the organ homogenates partly appear to depend on the feeding mode, but are rather characteristic of the fish variety.     

Comparative antioxidant enzyme study in freshwater fishes. I. Distribution of superoxide dismutase, peroxide-decomposing enzymes and lipid peroxidation in herbivorous fishes

The activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase were comparatively studied in organ homogenates from two herbivorous fishes, viz., the grass carp and the silver carp. 2. The protein contents and lipid peroxidation of organ homogenates were also compared. The comparative measurements primarily provide control values for subsequent toxicological examinations. 3. The highest total superoxide dismutase activities were found in the kidney, spleen and liver in the grass carp, and in the kidney and liver in the silver carp. 4. The antioxidant enzyme activities and other parameters of the organ homogenates appear to be independent of the feeding mode, but are rather characteristic of the fish variety.     

In vitro formation of organ specific proximate carcinogen of benzo(a)pyrene by rat homogenates

In order to determine the organ specific carcinogenicity of benzo(a)pyrene (B(a)P), its metabolites, formed in vitro by incubation with the homogenates from liver, lungs, kidneys, intestine and brain of rats, were isolated by TLC and spectroscopy. B(a)P was found to be converted into a number of metabolites by different tissue homogenates. The results showed that the proximate carcinogenic metabolite, 7,8-dihydro-7,8-dihydroxy B(a)P was formed only when rat lung and kidney homogenates were incubated with B(a)P in vitro. The UV spectral analysis also confirmed the formation of this metabolite only on incubation of B(a)P with rat lung and kidney homogenates. As the proximate carcinogenic metabolite was only formed by incubating B(a)P with the homogenates from target organs, its organ specific carcinogenicity may be explained.     

Efficient use of a combination of ampicillin and chymotrypsin]

The effect of chimotripsin on the level and duration of the ampicillin concentration increase in rats, as well as the effect of the enzyme on the in vitro antibiotic detection in the blood serum and organ homogenates of the animals was studied. It was found that rational combined use of ampicillin and chimotripsin required the enzyme administration not later than 1 hour before the antibiotic injection. Chimotripsin provided increased ampicillin levels in the blood serum and liver of the rats for at least 5 hours and in the kidneys and lungs for at least 4 hours. The enzyme present in the rats for 2 hours had no effect on determination of ampicillin activity in vitro in the presence of the blood serum and organ homogenates of the animals.     

Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

The Biological Reductive Capacity of Tissues is Decreased Following Exposure to Oxidative Stress: A Cyclic Voltammetry Study of Irradiated Rats

The reductive capacity of rat tissue homogenates and body fluids was determined by cyclic voltammetric measurements. The reductive capacity of rat lung, liver and kidney homogenates was significantly reduced four days after total body γ-ray irradiation with 5.5 Gy as compared to controls. In parallel, reduced ability of the irradiated organ homogenates to scavenge hydroxyl radicals and to destroy hydrogen peroxide was recorded. However, no difference in their superoxide dismutase activity was found. The possible use of cyclic voltammetry as a method for qualitative evaluation of the ability of biological tissues to cope with oxidative stress is discussed.     

Peroxidation of biomembrane lipids and its enzymatic regulation during aging in the rat

Measurement of the intensity of chemiluminescence and accumulation of the peroxidation products detected in the reaction with thiobarbituric acid (TBA-active products) has shown that with the ageing of rats the intensity of peroxidation of biomembranes' lipids gets lower in the liver and heart homogenates and tends to decrease in the brain homogenates. A strict correlation is established to exist between the level of TBA-active products and the chemiluminescence intensity independent of the age, organ and the method of initiation of lipids' peroxidation and its intensity. Considerable age changes in the rate of lipids' peroxidation in the liver and heart homogenates may be associated with an increase in the activity of a number of such antioxidant enzymes as superoxide dismutase, glutathione peroxidase, glutathione transferase and catalase in the postnatal ontogenesis of rats.     

Bacteriological examination of specified-pathogen-free animals: a procedure for small rodents

Organs of 10 mice or 5 rats were pooled and homogenized. A portion of the homogenate was cultured on several selective and enrichment media. The method was tested by adding various pathogenic organisms to organ homogenates. 10(3)-10(4) microorganisms in homogenates of 10 organs were readily detected. This procedure allows even a small microbiological laboratory to detect a low frequency of infected animals in a colony.     

The intrasanguineous host mediated assay procedure using Saccharomyces cerevisiae: comparison with two other metabolic activation systems

3 metabolic activation systems--organ homogenates, perfused liver, and the intrasanguineous host mediated assay (IHMA)--were compared in their abilities to activate demethylnitrosamine (DMN) and induce gene conversion in Saccharomyces cerevisiae D4. Both rats and mice were used for the organ homogenates and IHMA studies. All activation systems were able to activate DMN; where the different organs were compared, liver was more active than kidney, followed by lung. The IHMA was the most sensitive of the systems examined.     

A comparative study of some more important experimental animal peroxide metabolism enzymes.

1. We have studied and compared the peroxide metabolism enzymes (SOD, P and C) of the main organs of fresh-water mollusc, chicken, mouse, guinea-pig, rabbit, cat and dog. 2. The liver exhibited the highest SOD activity. The enzymatic activities of the organ homogenates of the guinea-pig stand out in comparison with the values for the other homogenates examined. 3. The liver, kidney and total brain homogenates of the chicken and the vertebrates display no, or only a very low P activity. The highest P activities were measured in the haemolysates. 4. The C and SOD values exhibit a certain parallelism. 5. The peroxide metabolism enzyme activities calculated by utilizing the protein measurements permit the establishment of a more realistic enzymatic activity.     

Cytochrome content in the liver of rabbits in chronic carbon tetrachloride poisoning after perenteral administration of exogenous cytochrome C]

Administration of exogenous cytochrome C to rats poisoned with CCl4 prevented reduction of the cytochrome C content in the homogenates, and of cytochromes a+a3, b and c+c1 in the hepatic mitochondria; it also promoted the normalization of the histostructure of the organ.     

Comparative study of the interaction of cephalosporins with blood serum proteins and organ homogenates

Interaction of 7 semisynthetic antibiotics (cephaloridine, cephalexin, cephradine, cephazolin, cephalotin, cephacetrile and cephapirin) with proteins of human, bovine and rabbit blood serum, as well as organ and tissue homogenates of rats was studied comparatively. The study showed that binding of the cephalosporins by the blood serum depended on both the chemical structure of the antibiotic and the species affiliation of the protein substrate. The binding lvels of cephazolin and cephalotin by the blood serum proteins (except bovine serum) were the highest, while the binding level of cephaloridine was the lowest. A significant decrease in the values of binding by the serum proteins of the drugs with high percentage of binding was observed when the drug concentrations in solution were increased. Binding of the cephalosporins by the blood serum proteins was in most cases completely reversible. The activity of the cephalosporins decreased in the presence of the rat organ and tissue homogenates. The levels of the activity decrease as compared to the theoretical ones were the highest with the use of cephalotin, cephacetrile and cephapirin. The lowest values of detection of these antibiotics were noted on their incubation with the liver, kidneys and lungs.     

Organ specific expression of esterase-6 in the house mouse,Mus musculus

Summary Esterase-6 in fresh homogenates of heart muscle and testis of the house mouse shows a two band (C allele) or three band (A allele) pattern in disc electrophoresis. These primary bands generated a series of secondary bands upon lowering the pH of the homogenates, and the secondary pattern, possibly resulting from partial proteolysis, was seen in varying degrees in fresh homogenates from a range of organs. Interrelationships between the primary and secondary bands were demonstrated by isoelectric focusing. The esterase-6 content of twenty different organ homogenates was estimated from electrophoretic gels, and a high level of this enzyme was observed in those organs most actively involved in fat metabolism. The possible participation of esterase-6 in fatty acid utilization is discussed. Similarities between esterase-6 of the house mouse and esterase-4 of the rat were demonstrated, further strengthening the view that these enzymes are homologous.Supported by the Deutsche Forschungsgemeinschaft (SFB 46)This is communication no 36 of a research program devoted to the cellular distribution, genetics, and regulation of non-specific esterases     

Effect of amphotericin B and its combination with 5-fluorouracil on the development of transplantable rat lymphosarcoma]

The studies were carried out on rats with subcutaneously transplanted metastizing Pliss lymphosarcoma. The animals were treated with amphotericin B administered intravenously in complex with sodium desoxycholate in doses of 336 and 20 Units/kg for 7 days beginning from 24th hour after the tumor transplantation. The treatment resulted in a statistically reliable inhibition of the tumor metastasizing as compared to the control animals. When the animals with the tumors were subjected to combined therapy with 5-fluoruracyl and amphotericin B, the antitumor and antimetastatic effect of 5-fluoruracyl significantly increased. Determination of amphotericin B binding with the organ homogenates showed that the antibiotic bound with the homogenates or the organs of the rats with the tumors to a greater extent than with those of the intact animals. The levels of the antibiotics in the tumor metastases were higher than those in the primary tumors.     

Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo.

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor kappaB (NF-kappaB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

In vitro metabolism of bladder carcinogenic nitrosamines by rat liver and urothelial cells.

In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell alpha-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybutyl)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37 degrees C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the alpha-hydroxylation pathway. Using hepatic S9 fractions, 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 +/- 0.02 nmol/h per mg protein (mean +/- S.E.) to 5.78 +/- 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N2 produced by urothelial cell homogenates was about 40-50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the alpha-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N2 production was about 40-50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.     

Effects of AET and MEA on the lysosomal hydrolase activities in the mouse organs.

The adult male Swiss mice were injected intraperitoneally with AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl), in a toxic dose of 400 mg/kg body weight. The acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E. C. 3.1.6.1) activities in crude homogenates of liver and kidneys were assessed every fourth hour throughout a 24-h period. Different patterns of temporal changes in the acid phosphatase and arylsulphatase activities in liver and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue and per the whole organ weight, were found. The extent and timing of the alterations in the activity of each of the lysosomal hydrolases were dependent on the particular organ chosen and aminothiol compound given.     

Binding of cationized and native ferritin to cellular structures of the electric organ of Torpedo marmorata

The distribution of polycationic and polyanionic binding sites in the electric organ of Torpedo marmorata was investigated by incubation of tissue with native (NF) ferritin. 1) Collagen fibrils from the electric organ carry rosettes of polyanionic sites on their surface with a periodicity of 60 nm, corresponding to the pattern of crossbanding in collagen fibrils. The CF-binding sites are abut 30 nm in size and project 20 nm beyond the surface of the fibril. 2) As revealed by incubation of tissue homogenates, CF heavily stains the intraperiod line of the axonal myelin and also tubular structures in the axonal cytoplasm. 3) Neither the extracellular aspects of the pre- nor the postsynaptic membrane became labeled with either NF or CF. After incubation of tissue homogenates. labeling of the electron-dense material of the cytoplasmic aspect of the postsynaptic membrane was observed with NF and, in particular, with CF. The ventral basal lamina of the electroplaque cell revealed uniform labeling with NF. In contrast, CF-binding sites were distributed in the lamina densa of the basal lamina as a lattice of discrete binding sites, approximately 45 nm in diameter. The presence of polyanionic sites in the basal lamina, which also proceeds through the synaptic cleft, suggests the existence of a diffusion barrier for the released neurotransmitter acetylcholine. It is proposed that this facilitates hydrolysis of acetylcholine in the synaptic cleft and recirculation of the products of hydrolysis to the axon terminal.     

The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We (34) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.