Saturated C21-C33 hydrocarbons are involved in the self-regulation of Pseudomonas fluorescens adhesion to a glass surface

One of the two putative groups of antiadhesions was identified in Pseudomonas fluorescens by the method of gas chromatography-mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10-15 micrograms/l; the concentrations of individual HC ranged from 0.1 to 3.0 micrograms/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces.

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.     

Variability of the Influence of Physicochemical Factors Affecting Bacterial Adhesion to Polystyrene Substrata

The role of electrostatic and hydrophobic interactions and solid and liquid surface tensions in the adhesion of four bacterial species (Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp., and Flexibacter sp.) to hydrophobic polystyrene petri dishes and to more hydrophilic polystyrene tissue culture dishes was investigated. The effect of electrostatic interactions was investigated by determining the effects of different electrolyte solutions on attachment to and of different electrolyte and pH solutions on detachment from the polystyrene substrate. The significance of solid and liquid surface tensions and hydrophobic interactions was investigated by measuring the effects of different surfactants (including a concentration series of dimethyl sulfoxide) on adhesion and detachment. Adhesion varied with bacterial species, substratum, and electrolyte type and concentration, with no apparent correlation between adhesion and electrolyte valence or concentration. The influence of different pH and detergent solutions on bacterial detachment also varied with species, substratum, pH, and detergent type; however, the greatest degree of detachment of all strains from the surfaces was produced by detergent treatment. The results suggest that adhesion cannot be attributed to any one type of adhesive interaction. There was some evidence for both electrostatic and hydrophobic interactions, but neither interaction could wholly account for the data.     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Cadmium biosorption properties of the metal‐resistant Ochrobactrum cytisi Azn6.2

The aim of this work was to establish the conditions for using Ochrobactrum cytisi Azn6.2 as a metal biosorbent. Azn6.2 is a novel strain from the legume symbiont O. cytisi that has been isolated from nodules of Medicago polymorpha plants grown on heavy metal‐polluted soils. Compared with the strain ESC1, Azn6.2 showed some biochemical differences, as well as antibiotic susceptibility, Azn6.2 was multi‐resistant to heavy metals, such as Cu, Cd and Zn, and bacterial pellets were able to biosorb high amounts of Cd and Zn. As shown by scanning electron microscopy coupled to energy dispersive X‐ray, most of Cd was attached to the cell surface. Optimal conditions for Cd biosorption were established, being 1 mM Cd ions in solution and 2 h of contact with the biosorbent at room temperature. At these conditions, maximal Cd loading capacity reached 32–34 mg/g. Cd desorption from bacterial pellets was achieved after washing with EDTA or, at higher efficiency, at pH 1.0. These results indicated that biosorption/desorption on O. cytisi Azn6.2 biomass should be a cost‐effective method for Cd recovery from contaminated solutions.     

Modeling the rate of attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the probability of their detachment from, potato tissue at 10 degrees C

The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions. Attachment was rapid and reached equilibrium after contact for 60 min. A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for Pseudomonas fluorescens remained unchanged for contact times between less than 5 s and 60 min. Listeria monocytogenes, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for Pseudomonas fluorescens thereafter. For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79.     

Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum

We assessed the degree to which Cd, Cr and Zn bound with sediment were assimilated by the green mussel Perna viridis and the Manila clam Ruditapes philippinarum. The influences of the metal concentration in the sediment, the presence of phytoplankton, and the oxidation condition of the sediment on metal assimilation were examined. No major difference was found for metal assimilation efficiency (AE) in sediment with different metal concentrations, except for Cd in the green mussels, in which the AE increased by 1.7x when the Cd concentration in sediment was elevated to 15x the natural background level. The higher assimilation of Cd with increasing Cd load in ingested sediment may be due to the higher desorption of Cd in the acidic gut of the bivalves. Both mussels and clams assimilated metals at a higher efficiency from the diatom diet (Thalassiosira pseudonana) than from inorganic sediment particles. The presence of algal particles had little influence on metal assimilation from ingested sediment, and conversely, the presence of sedimentary particles had little effect on metal assimilation from ingested diatom (except for Cd in the mussels). In the mussels, AEs were higher from oxic sediment than from anoxic sediment by 3.1x for Cd, 2.0x for Cr, and 1.4x for Zn, and in the clams AEs were higher from oxic sediment by 2.8x for Cd, 2.0x for Cr, and 2.0x for Zn. Our study suggested that metals associated with anoxic sediment can be potentially available to marine bivalves, and that metal AEs determined for a single diet were probably not affected by the presence of other food particles.     

Effect of surface shear stress on the attachment of Pseudomonas fluorescens to stainless steel under defined flow conditions

The application of the radial-flow growth chamber to the study of the initial stages of bacterial adhesion to surfaces under flowing conditions is reported. The adhesive properties of the bacterium Pseudomonas fluorescens (NCIB 9046) to stainless steel (type AISI 316) were found to be highly dependent on surface shear stress and the time and concentration of cells used in the incubation procedure. Maximum levels of adhesion occurred in zones of lowest surface shear stress, particularly less than 6-8 Nm(-2). Adhesion was still noticeable at shear stresses even up to 130 Nm(-2). Significant detachment of cells from a monolayer attached under static conditions was found to occur at surface shear stresses in excess of 10-12 Nm(-2).     

In situ monitoring of the nascent Pseudomonas fluorescens biofilm response to variations in the dissolved organic carbon level in low-nutrient water by attenuated total reflectance-Fourier transform infrared spectroscopy

Drinking water quality management requires early warning tools which enable water supply companies to detect quickly and to forecast degradation of the microbial quality of drinking water during its transport throughout distribution systems. This study evaluated the feasibility of assessing, in real time, drinking water biostability by monitoring in situ the evolution of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) fingerprint of a nascent reference biofilm exposed to water being tested. For this purpose, the responses of nascent Pseudomonas fluorescens biofilms to variations in the dissolved organic carbon (DOC) level in tap water were monitored in situ and in real time by ATR-FTIR spectroscopy. Nascent P. fluorescens biofilms consisting of a monolayer of bacteria were formed on the germanium crystal of an ATR flowthrough cell by pumping bacterial suspensions in Luria-Bertani (LB) medium through the cell. Then they were exposed to a continuous flow of dechlorinated sterile tap water supplemented with appropriate amounts of sterile LB medium to obtain DOC concentrations ranging from 1.5 to 11.8 mg/liter. The time evolution of infrared bands related to proteins, polysaccharides, and nucleic acids clearly showed that changes in the DOC concentration resulted in changes in the nascent biofilm ATR-FTIR fingerprint within 2 h after exposure of the biofilm to the water being tested. The initial bacterial attachment, biofilm detachment, and regrowth kinetics determined from changes in the areas of bands associated with proteins and polysaccharides were directly dependent on the DOC level. Furthermore, they were consistent with bacterial adhesion or growth kinetic models and extracellular polymeric substance overproduction or starvation-dependent detachment mechanisms.     

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.     

Biosorption of nickel and cadmium by metal resistant bacterial isolates from agricultural soil irrigated with industrial wastewater

Agricultural soil irrigated with industrial wastewater (more than two decades) analysed for heavy metals revealed high levels of Fe, Cr, Cu, Zn, Ni and Cd. Out of a total of 40 bacterial isolates obtained from these soils, 17 belonged to the family enterobacteriaceae and 10 were Pseudomonas spp. A maximum MIC of 200 for Cd, 400 for Zn and Cu, 800 for Ni, and 1600 microg/ml for Pb was observed. Biosorption of Ni and Cd studies over a range of metal ion concentrations with Escherichia coli WS11 both in single and bi-metal systems showed that the adsorption of Cd and Ni was dependent on the concentrations and followed the Freundlich adsorption isotherm. The biosorption of Ni increased from 6.96 to 55.31 mg/g of cells, and Cd from 4.96 to 45.37 mg/g of cells at a concentration ranging from 50 to 400 microg/ml after 2h of incubation in a single metal solution. A further increase in incubation time had no significant effect on the biosorption of metals.     

Development of Metal Tolerance in Soil Bacterial Communities Exposed to Experimentally Increased Metal Levels

The development of metal tolerance in soil bacterial communities exposed to different heavy metals was examined under laboratory conditions. An agricultural soil amended with different Zn concentrations was studied most intensively, and measurements were made over a 28-month incubation period by means of the thymidine incorporation technique. Tolerance levels were not affected by metal concentrations lower than 2 mmol of Zn kg (dry weight) of soil(sup-1), but above this value, the level of Zn tolerance increased exponentially with the logarithm of the soil Zn concentration. An increased metal tolerance was detected after only 2 days of Zn exposure. Thereafter, stable tolerance values were observed at different sampling times for bacterial communities exposed to up to 8 mmol of Zn kg (dry weight)(sup-1), indicating no changes in tolerance with time. The tolerance of bacterial communities exposed to 32 mmol of Zn kg (dry weight)(sup-1) increased rapidly within the second week of incubation, but then the values remained unchanged until the end of the experiment. Bacterial communities from soil contaminated with 16 mmol of Zn kg (dry weight)(sup-1) showed an increase of the same magnitude, but the increase started later, after 4 months of incubation, and took place for a much longer period (more than 1 year). Cd, Cu, and Ni addition also resulted in metal-tolerant communities, and the level of tolerance increased with prolonged incubations of the soils. The bacterial community at the end of the incubation period also exhibited a lower pH optimum and an increased tolerance to low osmotic potential. The results suggest that the increase in metal tolerance of the community after adding metals can be attributed to an immediate effect due to the death of sensitive species and a later effect due to different competitive abilities and adaptation of surviving bacteria.     

Oxidative Stress Induced by Cadmium in the C6 Cell Line: Role of Copper and Zinc

In this report, we have investigated the role of copper (Cu) and zinc (Zn) in oxidative stress induced by cadmium (Cd) in C6 cells. Cells were exposed to 20 μM Cd, 500 μM Cu, and 450 μM Zn for 24 h. Then, toxic effects, cellular metals levels, oxidative stress parameters, cell death, as well as DNA damage were evaluated. Cd induced an increase in cellular Cd, Cu, and Zn levels. This results not only in the inhibition of GSH-Px, GRase, CAT, and SOD activities but also in ROS overproduction, oxidative damage, and apoptotic cell death not related to Cu and Zn mechanisms. The thiol groups and GSH levels decreased, whereas the lipid peroxidation and DNA damage increased. The toxicity of Zn results from the imbalance between the inhibition of antioxidant activities and the induction of MT synthesis. The increase in Cu and Zn levels could be explained by the disruption of specific transporter activities, Cd interference with signaling pathways, and metal displacement. Our results suggest that the alteration of Cu and Zn homeostasis is involved in the oxidative stress induced by Cd.     

Effects of zinc and cadmium on erythrocyte antioxidant systems of a freshwater fish Oreochromis niloticus

In this work to determine the effects of metals exposure of Oreochromis niloticus on erythrocyte antioxidant systems, fish were exposed to 5.0 mg/L Zn, 1.0 mg/L Cd, and 5.0 mg/L Zn + 1.0 mg/L Cd mixtures for 7 and 14 days and reduced glutathione (GSH) level, catalase (CAT), and glucose‐6‐phosphate dehydrogenase (G6PD) activities were investigated. In addition, Zn or Cd levels in whole blood were studied. Erythrocyte GSH level and CAT and G6PD enzyme activities increased in response to single and combined Zn and Cd exposure. The elevation observed in the CAT activity was higher in the Cd alone, and in combination with Zn, than in Zn alone. Time‐dependent alteration was not observed in all antioxidant parameters. Exposure to metals (alone and in mixture) resulted in elevatation of Zn and Cd levels in the blood. Concentration of metals in the blood of fish exposed to the Zn + Cd combination was lower than in fish exposed to the single metal. This study demonstrates that metals caused oxidative stress in fish erythrocytes, and an adaptation with an increase in CAT and G6PD activities and GSH level, which were important in the protection against metal damage, was observed. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:223–229, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20327     

Effects of cadmium, copper, magnesium, and zinc on the decomposition of citrate by a Klebsiella sp

The effects of Cd2+, Cu2+, Mg2+, and Zn2+ on the decomposition of citric acid by a Klebsiella sp. were studied by monitoring the degradation of [14C]citrate. The carbon concentration used was 10 micrograms of C liter-1, and the media were designed to provide at least 95% of the citrate complexed to the metal studied. After 72 h of incubation, 80% of the uncomplexed citric acid and 76% of the magnesium citrate had been decomposed. A marked inhibition was observed when Cd2+, Cu2+, or Zn2+ was bound to the organic anion; only 23% of the cadmium citrate, 14% of the zinc citrate, and 5% of the cuprous citrate had been decomposed. The effects were not the result of toxicity, since experiments run with [14C]glucose (nonchelating compound) instead of citrate resulted in similar decomposition rates regardless of the presence of the metal. To examine whether the binding of a metal to citrate enhanced its uptake by the Klebsiella sp., we studied the relative uptake of 65Zn in citrate- and in glucose-containing media. No such effect could be observed, with the uptake of Zn2+ being higher in the glucose-containing media. The study shows that metals may render low-molecular-weight organic acids, such as citric acid, resistant to bacterial degradation. This stresses the importance of metals in influencing microbial decomposition of organic compounds, not only as a result of toxicity.     

Effects of cadmium, copper, magnesium, and zinc on the decomposition of citrate by a Klebsiella sp.

The effects of Cd2+, Cu2+, Mg2+, and Zn2+ on the decomposition of citric acid by a Klebsiella sp. were studied by monitoring the degradation of [14C]citrate. The carbon concentration used was 10 micrograms of C liter-1, and the media were designed to provide at least 95% of the citrate complexed to the metal studied. After 72 h of incubation, 80% of the uncomplexed citric acid and 76% of the magnesium citrate had been decomposed. A marked inhibition was observed when Cd2+, Cu2+, or Zn2+ was bound to the organic anion; only 23% of the cadmium citrate, 14% of the zinc citrate, and 5% of the cuprous citrate had been decomposed. The effects were not the result of toxicity, since experiments run with [14C]glucose (nonchelating compound) instead of citrate resulted in similar decomposition rates regardless of the presence of the metal. To examine whether the binding of a metal to citrate enhanced its uptake by the Klebsiella sp., we studied the relative uptake of 65Zn in citrate- and in glucose-containing media. No such effect could be observed, with the uptake of Zn2+ being higher in the glucose-containing media. The study shows that metals may render low-molecular-weight organic acids, such as citric acid, resistant to bacterial degradation. This stresses the importance of metals in influencing microbial decomposition of organic compounds, not only as a result of toxicity.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Effects of dexamethasone on metallothionein induction by Zn, Cu, and Cd in Chang liver cells

Metallothioneins (MTs) were induced in Chang liver cells by the metals, Zn, Cu and Cd, and the glucocorticoid hormone, dexamethasone. When 116 microM Zn, 32 microM Cu and 18 microM Cd, and 10(-7) M dexamethasone, respectively, were administered for 9 h, MTs induced by each inducer in the cells reached maximum levels. The maximum accumulation of MT level induced by dexamethasone was the lowest of the four inducers investigated; the levels induced by Zn, Cu and Cd were 4.7, 1.2 and 1.5 times of that induced by dexamethasone. When dexamethasone was added to the cells together with the heavy metals (Zn, Cu and Cd), dexamethasone had an additive effect on the maximum MT accumulations induced by heavy metals as compared to when induction was conducted using one of heavy metals alone or by dexamethasone alone. However, dexamethasone did almost not effect the metal accumulations in the cells, although the maximum MT levels induced by heavy metal increased by dexamethasone. These results suggest that the process of MT induction by heavy metals and that by dexamethasone are independent of one another. When dexamethasone was added to the cells together with a high concentration of Cu (32 microM) induced the maximum MT accumulation, Cu transport into the cells decreased by 20-40% of that into non-treated cells, which was statistically significant.