Immunological and cytotoxicological responses of the Asian clam, Corbicula fluminea (M.), experimentally exposed to cadmium

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^-1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^-1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^-1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.     

Immunological and cytotoxicological responses of the Asian clam,Corbicula fluminea (M.), experimentally exposed to cadmium

Abstract

Bivalve molluscs, as filter-feeding organisms, are known to accumulate metals that can produce deleterious effects on organisms. The phagocytic activity of haemocytes and lysosomal alterations in the digestive gland cells were measured in the freshwater Asian clam exposed to cadmium, in order to assess the possible use of immunocompetence and lysosomal responses as biomarkers of freshwater quality. Clams were exposed in the laboratory to nominal concentrations of 3, 10, 21.4, 46.5 and 100 µg l^?1 of cadmium and sampled after 7, 15 and 30 days of exposure. The results show a decrease of phagocytic activity after only 7 days of exposure to 10 µg l^?1 of cadmium. This response was also observed as the exposure time was increased. Lysosomes in the digestive cells increased in size and number after 7 days of exposure as cadmium concentration increased. After 30 days of exposure, a decrease in size and number indicated a change in the response to the metal from concentrations of 46.5 µg l^?1 of cadmium. A dose and time response both in phagocytic activity of haemocytes and lysosomal structure demonstrated a possible use of these biomarkers in freshwater biomonitoring.     

Cadmium removal by living cells of the marine microalga Tetraselmis suecica

Cadmium removal by living cells of the marine microalga Tetraselmis suecica was tested in cultures exposed to different cadmium concentrations (0.6, 3, 6, 15, 30 and 45 mg/l). The EC50 for growth was 7.9 mg Cd/l after six days of exposure. The cadmium removed was proportional to the concentration of this metal in the medium and it was dependent on the time of exposure; cultures with higher cadmium concentration removed a higher amount of this metal. In cultures exposed to 0.6 mg/l, T suecica cells removed 98.1% of added cadmium with 0.392 x 10(-6) microg Cd/cell, whereas in cultures with 45 mg/l only 7.7% was removed with 16.052 x 10(-6) microg Cd/cell. The highest amount of cadmium removed per liter of culture was observed in cultures exposed to 6 mg/l, with 3.577 mg/l of cadmium. After six days of incubation, the higher proportion of cadmium was bioaccumulated intracellularly in all cultures except in 45 mg/l cultures, the percentage of intracellular cadmium being always more than 50%. The highest percentage of bioadsorbed cadmium (60.1%) was found in cells of cultures with the highest cadmium concentration (45 mg/l). Furthermore, a relation between intracellular cadmium and the concentration of sulfhydryl groups was observed.     

A comparison of the differential accumulation of cadmium in the tissues of three species of freshwater fish, Salmo gairdneri, Rutilus rutilus and Noemacheilus barbatulus

Roach and stone loach were exposed to cadmium dissolved in their aquarium water at 500 and 1250 micrograms/l, respectively, and the distribution of the metal accumulated in the major body organs was determined. The pattern of distribution for each species was somewhat different and was distinct in each case from that observed previously with rainbow trout. The total body loads of cadmium accumulated by the three species were assessed during the period of exposure and found not to correlate directly with the concentration of cadmium to which the individual species had been exposed. An alternative comparator was devised which as the quotient of the total body cadmium accumulation (microgram/100 g body wt) and the notional cadmium dose (microgram/l) X weeks was described as a fractional retention coefficient for cadmium. The coefficient was constant for each species at different periods of exposure to cadmium alone. The values of the coefficient for roach and stone loach were however much lower than that for rainbow trout. When rainbow trout were preexposed to zinc (100 micrograms/l, 5 days) before being exposed to cadmium, the fractional retention coefficient for cadmium fell to a value similar to those seen with roach and stone loach exposed to cadmium alone. The significance of these observations in relation to the nature of the intracellular proteins to which cadmium is bound in the three species is discussed in the light of their differential susceptibility to the toxic effects of cadmium.     

Effect of the surfactant Corexit 7664 on uptake of cadmium by organisms and biological matter in a closed circulated brackish-water system

Juvenile flounder and common mussel from the Baltic Sea were kept in Corexit 7664 (50 µg/l) and cadmium (5 µg/l)-contaminated, recirculated, sea-water systems for 200 days at 15 °C and 20 S. Accumulation of cadmium with exposure time was measured in several fish tissues and in mussel. There were no differences in the cadmium accumulation by tissues with or without the addition of Corexit 7664. No acute effects of the contaminants on the experimental animals could be noted. Final cadmium concentrations, reached for all biological matter analysed, were 4 times higher than in full-strength sea water.     

The effects of cadmium on ALA-D activity, growth and haemoglobin content in the water flea, Daphnia magna

The ALA-D activity, haemoglobin content and growth was studied in the water flea, Daphnia magna, exposed to 0, 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms Cd/l. The ALA-D activity in water fleas exposed to 0.2-1.6 micrograms Cd/l fluctuated around the control value. The activity in animals exposed to 0.1 micrograms Cd/l decreased during the entire experiment. After 16 days exposure to cadmium the haemoglobin content in water fleas ranged between 80 and 31% of control value. In animals exposed to 0.8 and 1.6 micrograms Cd/l the haemoglobin content decreased progressively during the experiment. Growth was not affected by cadmium at these concentrations.     

Activities of superoxide dismutase and catalase in erythrocytes and plasma transaminases of goldfish (Carassius auratus gibelio Bloch.) exposed to cadmium

Four groups of goldfish were exposed to cadmium in a concentration of 20 mg Cd/l water under aquarium conditions. The duration of exposure was 1, 4, 7 and 15 days. It was shown that the activity of superoxide dismutase (SOD) in the red blood cells (RBC) significantly decreased after the first day of cadmium exposure. However, the SOD activity increased after 7 and 15 days of cadmium treatment. Elevated activity of catalase (CAT) was found in erythrocytes of cadmium-treated fishes after 15 days, whereas plasma GOT levels was increased after 7 and 15 days and GPT levels after 1, 4, 7 and 15 days of cadmium treatment. This was accompanied by a significant decrease of blood hemoglobin concentrations (after 15 days) and hematocrit values (after 7 and 15 days). However, the concentration of blood glucose significantly increased after 1, 4, 7 and 15 days of cadmium exposure. These results indicate that cadmium causes oxidative stress and tissue damage in the exposed fishes.     

The effects of heavy metals on common carp white blood cells in vitro

The in vitro effects of cadmium, copper, lead and zinc, and various cadmium compounds (chloride, sulphate and nitrate) on common carp (Cyprinus carpio) lymphocyte viability and phagocyte activity, were evaluated. The percentage of dead lymphocytes was determined after Trypan blue staining, and phagocyte activity was measured by using the nitroblue tetrazolium (NBT) reduction test. Lead was the most toxic to lymphocytes--the maximum mortality exceeded 30%, and was significantly higher at 1 microM of lead, compared to the control. The maximum mortality caused by cadmium was below 10%, but was significantly elevated with 5 microM or more of cadmium. Zinc induced lymphocyte mortality from 10 microM, whilst no effect was observed with copper. The incubation of full blood with the three cadmium compounds (at 5mg/l of cadmium) showed that cadmium nitrate and cadmium sulphate were more toxic (over 35% and 25% mortality, respectively) than cadmium chloride (about 15% mortality). This was confirmed by the results of tests on isolated cells--1mg/l of cadmium as nitrate and sulphate increased lymphocyte mortality compared to the control and cadmium chloride. Phagocytic activity was less sensitive to heavy metals than was lymphocyte viability. It was significantly reduced following exposure to 50 microM and 100 microM cadmium, and 100 microM zinc, but no effects were observed with either lead or copper.     

Disinfectant effects of hot water, ultraviolet light, silver ions and chlorine on strains of Legionella and nontuberculous mycobacteria

The disinfectant effects on Legionella and nontuberculous mycobacteria of hot water, ultraviolet light, silver ions and chlorine, were evaluated. The bacterial strains Legionella pneumophila ATCC33152 and Mycobacterium avium ATCC25291 and strains of L. pneumophila and M. avium which had been isolated from a 24 h bath, were examined for their resistance to treatments. All strains were killed within 3 min on exposure to hot water at 70 degrees C and exposure to ultraviolet light at 90 mW.s/cm2. The strains of L. pneumophila tested were killed within 6 h on exposure to a solution of silver ions at 50 micrograms/l. The number of viable cells of strains of M. avium fell from 10(5) CFU/ml to 10(3) CFU/ml after exposure to an aqueous solution of silver ions at 100 micrograms/l for 24 h. Chlorine effectively killed strains of Legionella which were exposed to an aqueous solution of chlorine at 2 mg/l within 3 min, but strains of Mycobacterium survived exposure to chlorine at 4 mg/l for more than 60 min.     

Effect of cadmium on the growth of Chlorella vulgaris and Stichococcus bacillaris

The growth of Chlorella vulgaris and Stichococcus bacillaris cultures in media containing from 20 to 100 mg Cd/l was studied. The examined strains were found to be highly resistant to the action of cadmium since the highest concentration of the metal used limited the production of dry weight (during 5 days of cultivation) by less than 50%. The lower production of chlorophyll a by S. bacillaris cultures in media containing from 60 to 100 mg Cd/l and 2-fold elongation of the cells point to lower tolerance of the strain to cadmium than that shown by C. vulgaris.     

Physiological, anatomic and behavioural changes in the respiratory system of Salmo gairdneri Rich. On acute and chronic exposure to chlorothalonil

1. Various changes in anatomical, physiological and behavioural aspects of the respiratory system of Salmo gairdneri on exposure to chlorothalonil (TCIN) were examined, along with the relationship between LC50 response and oxygen concentration. 2. The TCIN LC50 was significantly lower at 5 mg/l oxygen concentration than at 8 mg/l. 3. Haematocrit was depressed on exposure to TCIN, whereas exposure to pentachlorophenol and lindane raised the haematocrit. Muscle lactate was not elevated in acutely exposed fish. 4. Ventilatory frequency was raised above 30 micrograms/l TCIN. 5. Gills of fish exposed to 2.0 micrograms/l TCIN for 24 days had reduced lamellar diffusive capacity, primarily caused by an increase in blood barrier thickness.     

Cadmium accumulation in tissues of the mussel Mytilus galloprovincialis

Studies have been made on cadmium accumulation in tissues of mussels kept within 20-60 days in water artificially enriched by Cd up to 20-100 micrograms/l. Irrespectively of cadmium concentration in the medium, its accumulation in tissues decreases in the following order: mid-gut gland, gills, gonads, mantle, adductor. Maximum concentration of Cd was found in the digestive tubuli of the mid-gut gland by X-ray microanalysis. The increase in S and, to a lower extent, P concentrations in these tubuli was also observed. It is suggested that the latter is due to immobilization of Cd by metal-binding proteins as well as to lyzosomal vesicles involved into detoxication of Cd. The increase in the external cadmium up to 100 micrograms/l did not affect the level of K, Ca and Mg in tissues of the mussel.     

A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.     

Sequestration of environmental cadmium by metallothionein in the roach (Rutilus rutilus) and the stone loach (Noemacheilus barbatulus)

Two species of coarse fish that are relatively resistant to cadmium poisoning were exposed to sub-lethal concentrations of the metal in their aquarium water. Thus, roach were exposed to cadmium concentrations between 30 and 500 micrograms/l for periods of 14-70 days whereas stone loach were exposed to 1250 micrograms Cd/l for 21-77 days. Under all conditions of exposure, it was found upon analysis of the major organs of accumulation of cadmium in the two species that the toxic metal was sequestered by a single isoform of metallothionein. The amino acid compositions of roach and stone loach metallothionein were determined and found to be similar to those reported for other piscine metallothioneins. The two proteins were found to contain Cd:Zn:Cu in approximate ratios of 4:1:2 per mole of protein. The sequestration of Cd by metallothionein in the two resistant species of fish is contrasted with the situation observed previously in a cadmium-sensitive species, the rainbow trout.     

Uptake and distribution of cadmium in the egg of the teleost, Oryzias latipes

Eggs of Oryzius latipes in the blastula stage were exposed to M/100 artificial sea water which contained cadmium at the concentrations of 0.1, 1.0, 10.0, 20.0 or 50.0 mg 1⁻¹. The 96 h TL₅₀, value for cadmium was estimated to be 20 5 mg 1⁻¹. When the eggs were incubated for 24 h in the M/100 sea water with 10.0 mg Cd 1⁻¹ and then rinsed in glycine buffer solution (pH; 2.0), the cadmium content of the egg decreased markedly. Cadmium levels were determined in parts of the embryonic body, the chorion and the yolk sac. The most cadmium was detected in the chorion (94.6%). Prolonged cadmium exposure revealed that most of the cadmium was absorbed by the chorion and little was detected in the embryonic body and the yolk sac.     

Effect of chronic cadmium exposure on locomotor behaviour of rats

Growing male rats were exposed to cadmium (Cd, 100 micrograms/kg, ip) for 51 days and the effect on the different components of locomotor behaviour was assessed on days 38, 46 and 51 of Cd exposure. Significant decrease in distance travelled, stereotypic time and movements, ambulatory time and vertical movements were observed in Cd-exposed rats, whereas the time of rest was increased. The number of entries into the inner as well as the outer squares and the total time spent in the inner squares of the floor area were significantly reduced. Results indicate that Cd exposure results in a general depression in all aspects of motor behaviour leading to decrease in gross locomotor activity. The involvement of an exaggerated emotional reactivity in the behavioural expression of the Cd-treated animals is also emphasized.     

The effect of chronic exposure to cadmium and gamma radiation at low doses on the genetic structures in mice

The DNA-protein cross-links (DPC) in mouse thymocytes and spleen lymphocytes, the number of abnormal sperm heads (ASH) and the number of micronuclei (MN) in normochromatic erythrocytes (NCE) of peripheral blood were studied in mice exposed to long-term low-intensity gamma radiation (0.072 cGy/days) and/or cadmium with drinking water (0.01 mg Cd2+/l) for 20, 40 and 80 days. The dependence of DPC level on the total dose (exposure time) of gamma radiation and/or cadmium is nonlinear. The maximal level of DPC in cells of lymphoid organs upon exposure to gamma radiation or cadmium was recorded on the 40-th day, and under combined exposure on the 20-th day of the experiment. The long-term exposure to cadmium or gamma radiation causes an increase in the ASH frequency. The increase in frequencies of MN in NCE and reciprocal translocations in spermatocytes was not found.     

Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla

Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation-evoked secretion by 80-90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 micrograms), nicotine (5 microM), muscarine (50 micrograms), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine- and K(+)-evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine-evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetylcholine-evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.