Changes in the kidney peroxidase activity in fish exposed to some industrial pollutants.

Fish under abnormal environmental conditions suffer from retarded growth and other physiological dysfunctions related to thyroxine deficiency. In teleosts lacking a definite thyroid gland the kidney plays a very important role in biosynthesis of the thyroidal hormone, in which peroxidase has an indirect action. Effects of some industrial pollutants and factory effluents on fish kidney peroxidase activity were recorded in Ophicephalus punctatus and Clarias batrachus. At concentrations of the pollutants at which 70-100% of the fish survive the exposure, peroxidase activity was greatly inhibited, indicating that even sub-lethal doses of toxicants may cause drastic changes in the physiological systems. The peroxidase activity increased well above the control levels at 5-h and 27-h exposures in some cases, but declined towards the end of the test.     

Proteomic analysis of blood cells in fish exposed to chemotherapeutics: evidence for long term effects

Proteomics technology are increasingly used in ecotoxicological studies to characterize and monitor biomarkers of exposure. The present study aims at identifying long term effects of malachite green (MG) exposure on the proteome of peripheral blood mononuclear cells (PBMC) from the Asian catfish, Pangasianodon hypophthalmus. A common (0.1 ppm) concentration for therapeutic treatment was applied twice with a 72 h interval. PBMC were collected directly at the end of the second bath of MG (T1) and after 1 month of decontamination (T2). Analytical 2D-DIGE gels were run and a total of 2551±364 spots were matched. Among them, MG induced significant changes in abundance of 116 spots with no recovery after one month of decontamination. Using LC-MS/MS and considering single identification per spot, we could identify 25 different proteins. Additionally, MG residues were measured in muscle and in blood indicating that leuco-MG has almost totally disappeared after one month of decontamination. This work highlights long term effects of MG treatment on the PBMC proteome from fish intended for human consumption.     

Functional activity of phagocyte blood cells in pulmonary tuberculosis

The characteristics of the functional activity of phagocyte blood cells in patients with destructive pulmonary tuberculosis caused by medicinal sensitive and medicinal resistant infective agents were studied. In the process of pulmonary tuberculosis, irrespective of the medicinal sensitivity of infective agents before and during treatment, a decrease in the phagocytic activity of neutrophil granulocytes and the level of the expression of Fcgamma- and C3b-receptors on monocytes with a simultaneous increase in the spontaneous production of oxygen metabolites in neutophils and a rise in the adsorptive capacity of monocytic cells were observed.     

Rodlet cells in the epidermis of fish exposed to stressors

Rodlet cells (RC) were detected in the epidermis of carp (Cyprinus carpio) and trout (Oncorhynchus mykiss) exposed to stressors (e.g. acid water, heavy metals, thermal elevation, polluted water of the Rhine river, water loaded with organic manure, and distilled water), as well as after wounding. These cells were not found in the epidemis of fish kept under control conditions, thus suggesting that the appearance of RC in the epidermis is stressor-related. Immature RC, resembling mucous cells, were found in deeper epidermal zones while mature RC were found close to and at the skin surface. The latter contained several rodlets embraced within a capsule-like structure composed of filaments. At the skin surface, RC opened the filamentous capsule at the cell apex and a cytoplasmic tuft, containing the rodlets, protruded outwards into the surrounding water. Structures which appeared to be free rodlets were also observed in the water, near the ridges of the pavement cells. Cytochemical and immunocytochemical study revealed that the rodlets contain alkaline phosphatase at their periphery and peroxidase activity at their cores. These enzymes have been previously reported in mucous cells of stressed fish. All together, the location of RC, their structure, cytochemistry and direction of migration suggest that these cells form part of the non-specific defence mechanism of the skin, and possibly other epithelial tissues.     

Haematological and histological changes in fish Heteropneustes fossilis exposed to pesticides from industrial waste water

The present study investigated the potential changes in hematologic parameters and histology of fish Heteropneustes fossilis when exposed to industrial waste water of different concentrations. The toxicant enter into the fish body from different rout, inhalation, dermal oral, and other several rout for entering in the fish body. The blood parameters of fish H. fossilis control group and treatment were investigated on 1st, 5th, 10th, and 20th days of experiment, hematologic variation was observed in RBC, WBC, MCV, MCH, and MCHC count. Histopathologic changes in liver, intestine, gill, muscle, and heart showed increasing degrees of damage in the tissues in correlation with the accumulation pattern of pesticides, while liver, intestine, gill, muscle, and heart of control group exhibited a normal architecture. Toxic effects of pesticides vary in different organs of the fish H. fossilis significant. The fish exposed to higher concentration showed uncoordinated alterations in behavioral responses, especially erratic and jerky swimming, physiologic, malformation, histologic, hematologic and biochemical changes, frequent surfacing and in gulping, mucus secretion, an increase in opercular movement, and copious secretion of mucus of all over the body.     

Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds

BACKGROUND: Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. METHODS: The present study evaluated chemical-induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microscopy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI). RESULTS: The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelov's solution was unable to detect early apoptosis. CONCLUSIONS: The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge.     

Oxygen-related processes in red blood cells exposed to tert-butyl hydroperoxide

The correlation between the oxidative processes in tert-butyl hydroperoxide (tBHP)-exposed red blood cells and the reactions of oxygen consumption and release were investigated. Red blood cell exposure to tBHP resulted in transient oxygen release followed by oxygen consumption. The oxygen release in red blood cells was associated with intracellular oxyhaemoglobin oxidation. The oxygen consumption proceeded in parallel with free radical generation, as registered by chemiluminescence, but not to membrane lipid peroxidation. The oxygen consumption was also observed in membrane-free haemolyzates. The order of the organic hydroperoxide-induced reaction of oxygen release with respect to the oxidant (tBHP) was estimated to be 0.9 +/- 0.1 and that of the oxygen consumption reaction was determined to be 2.4 +/- 0.2. The apparent activation energy values of the oxygen release and oxygen consumption were found to be 107.5 +/- 18.5 kJ/mol and 71.0 +/- 12.5 kJ/mol, respectively. The apparent pKa value for the functional group(s) regulating the cellular oxyHb interaction with the oxidant in tBHP-treated red blood cells was estimated to be 6.7 +/- 0.2 and corresponded to that of distal histidine protonation in haemoprotein. A strong dependence of tBHP-induced lipid peroxidation on the oxygen concentration in a red blood cell suspension was observed (P50 = 32 +/- 3 mmHg). This dependence correlated with the oxygen dissociation curve of cellular haemoglobin. The order of the membrane lipid peroxidation reaction with respect to oxygen was found to be 0.5 +/- 0.1. We can conclude that the intensity of the biochemical process of membrane lipid peroxidation in tBHP-exposed erythrocytes is controlled by small changes in such physiological parameters as the oxygen pressure and oxygen affinity of cellular haemoglobin. Neither GSH nor oxyhaemoglobin oxidation depended on oxygen pressure.     

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.     

Surfacing activity and food utilization in a tropical air-breathing fish exposed to different temperatures

Reared in tubular aquaria containing different depths of water (2.5, 5.0, 15.5, 31.0 and 40.0 cm), the obligatory air-breathing fish Ophiocephalus striatus (760 mg; 4.5 cm L) was forced to swim vertically a longer or shorter distance per surfacing. Interaction of temperature (17, 22, 27, 32 and 37°C) and aquarium depth reveals that surfacing frequency of the fish, fed ad libitum on Tilapia muscle, increased with increasing aquarium depth, but the increase was significant only at 27 and 32°C; in the starving series, the frequency was not depth-dependent at any temperature. Owing to the sustained surfacing activity and the consequent fatigue, the test individuals ‘hung’ to the surface for a definite period. Hanging frequency was temperature-dependent, but not a depth-dependent activity either in the starving or feeding series. At any temperature and aquarium depth, the feeding series hung more frequently than the starving series. Hanging duration increased from about 1 hr/day in either series at 17°C to 6 and 18 hr/day in the feeding and starving series at 37°C. At any tested temperature, distance swum by the feeding and starving series was a depth-dependent activity. The feeding series at 32°C exhibited the maximum swimming speed of 2 L/sec for 4.8 hr/day in the 40 cm depth. With increasing temperature and depth, feeding rate increased (from 24 to 225 g cal/g live fish/day); between 17 and 27°C, it was more a temperature-dependent activity. The highest rate (47 g cal / g/day) and efficiency (27%) of conversion were observed at 32°C; whereas the efficiency was depth-dependent, the rate was not. Oxygen uptake was a temperature-dependent activity; aquarium depth played a secondary role. Briefly, O. striatus in deeper aquaria consumed significantly more food and converted lesser, as it surfaced more frequently and swam longer distance, dissipating more energy on metabolism and swimming activity. Hence, culturing O. striatus in shallow waters at the optimum temperature of 32°C will be advantageous. This work was supported by the University Grants Commission's (New Delhi) grant to Dr. T. J. Pandian: Grant No. F. 23-210/75 (Sr II) for which appreciation is expressed.     

Inositol pentaphosphate in fish red blood cells

Inositol pentaphosphate (IP5) has long been characteristic of avian erythrocytes. We now report that this compound is also present in the red cells of two species of elasmobranch fishes, Squalus acanthias (spiny dogfish) and Narcacion nobiliana (torpedo ray). The mean concentration of I5 is 0.36 and 0.24 micromoles per ml of red cells respectively. ATP is the major organic phosphate in both fishes. 2.3-diphosphoglycerate is absent and GTP levels correspond approximately to the levels of I5.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

Radiation-induced structural alterations in fish red blood cells

The structural properties of gamma-irradiated fish red blood cells were studied using a spin labelling method. The gradient increase of lipid fluidity with the increasing gamma radiation doses was indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spin labels spectra. In turns, the spectra of maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated a modification of the internal proteins and the increased internal viscosity of red blood cells. The results encourage the conclusion that the increase in membrane fluidity may result from theernations in lipid-protein interactions rather than lipid peroxidation.     

Surfacing activity and food utilization in the air-breathing fish polyacanthus cupanus exposed to constant Po2

The influence of feeding on the surfacing frequency of the air-breathing fishPolyacanthus cupanus was investigated. Feeding increased the surfacing and swimming activities. Food conversion, maintenance requirements and energy spent in swimming and surfacing were measured under different feeding regimes.     

Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.