Direct and Indirect (Foodweb Mediated) Effects of Metal Exposure on the Growth of Yellow Perch (Perca flavescens): Implications for Ecological Risk Assessment

Yellow perch (YP) are metal tolerant fish that form large populations in many metal-impacted regions across Canada. While they are able to survive and reproduce successfully in environments with water and sediment metal concentrations that are toxic to many invertebrates, perch experience a suite of direct and indirect impacts. YP were studied in a series of Canadian Precambrian Shield lakes near Sudbury, Ontario, along a gradient of metal exposure downwind from metal smelters. In lakes at the high end of our exposure gradient, concentrations of metals (Cu, Ni, and Cd) in YP liver and kidney were well above levels seen in fish from reference lakes. Direct effects linked to metal-exposure were observed, ranging from effects at the cellular level, to effects in organs and tissues, individuals, and populations. In addition to direct or physiological effects, we also documented indirect, foodweb-mediated effects of metals on YP in the most contaminated lakes resulting from the elimination of metal-sensitive large benthic invertebrates and their replacement by small metal-tolerant taxa. The most common indication of such indirect effects on YP is severely stunted growth coupled with a high degree of zooplankton dependence throughout their life. Such indirect effects have important implications for ecological risk assessment (ERA) because they indicate that higher trophic levels may be functionally altered even though the functional groups that they depend on are present and abundant. Although the functional groups important to yellow perch can be abundant in metal-impacted lakes, their benthic communities are impoverished and this is strongly reflected in their size structure upon which energy transfer to higher trophic levels depends. Thus indirect (foodweb-mediated) effects can be important in situations where invertebrate size structure is impacted in such a way as to reduce the efficiency of energy transfer to higher trophic levels, and therefore measures should be taken to protect and/or restore large sensitive benthic species.     

Subcellular Distribution of Cadmium and Nickel in Chronically Exposed Wild Fish: Inferences Regarding Metal Detoxification Strategies and Implications for Setting Water Quality Guidelines for Dissolved Metals

The objective of this study was to investigate metal detoxification in chronically exposed juvenile yellow perch (YP: Perca flavescens) and to field test the commonly assumed threshold toxicity model. Fish were collected from lakes located along a cadmium (Cd) and nickel (Ni) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Hepatic metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of YP liver homogenates. Major proportions of hepatic Cd were found in the heat-stable cytosolic peptides and proteins fraction (HSP; including metallothioneins), whereas Ni was mainly found in the potentially metal-sensitive heat-denaturable proteins fraction (HDP). For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction or the organelle fraction did not occur. Metal detoxification was clearly incomplete and P. flavescens was subject to some metal-related stress, as evidenced notably by endocrine perturbations. Similar subcellular partitioning results were obtained when juvenile yellow perch were transferred from a reference lake to a Cd-contaminated lake and Cd accumulation was followed over time; there was no accumulation threshold below which Cd binding to the putative metal-sensitive fractions (HDP and organelles) did not occur. The presence of Cd and Ni in these fractions, even for low exposure concentrations and low hepatic accumulation, contradicts the threshold toxicity model that underpins metal toxicology theory and that is implicitly used in setting water quality guidelines for metals. Chronically exposed YP appear to have settled for a tradeoff between the cost of turning on their detoxification apparatus at full capacity, to completely suppress metal binding to metal-sensitive sites, and the alternative cost of allowing some binding of inappropriate metals to metal-sensitive sites.     

Changes in metallothionein concentrations in response to variation in natural factors (salinity, sex, weight) and metal contamination in crabs from a metal-rich estuary

Intermoult male and female crabs Pachygrapsus marmoratus and Carcinus maenas were sampled from three sites between the mouth and 25 km upstream in the Gironde, the most Cd-contaminated estuary in France, in order to study the relative importance of natural factors (salinity, sex, weight) and accumulated metal concentrations on metallothionein (MT) concentrations. In the two species studied, higher metal, total protein and MT concentrations were observed in the hepatopancreas than in the gills. In P. marmoratus, MT concentrations were mainly related to changes in the natural factors even if MT and Zn concentrations were positively correlated in the hepatopancreas whereas in C. maenas, the main relationships were with accumulated metal levels. In the case of the natural factors, the most important ones were weight in gills of both crab species, and salinity changes in both hepatopancreas and gills of P. marmoratus. Cd and Cu concentrations in both organs of the two species were inversely related to salinity. The same observation was found for Zn concentrations in C. maenas but not in P. marmoratus. In the hepatopancreas of both species, the highest total protein concentrations were found in crabs from the site with the highest salinity, whereas there were no such differences in the gills. It seems that changes in MT concentrations are linked more to changes in general protein metabolism than to changes in metal accumulation. Thus it was important to examine the storage of metals in other tissue compartments, particularly the insoluble fraction which includes mineral granules which is known to also contribute to trace metal detoxification in invertebrates. In the gills of the crabs, Zn was present mainly in the insoluble fraction, whereas Cd was nearly equally distributed between soluble and insoluble fractions. In contrast, Cu in the gills and all three metals in the hepatopancreas of both species were mainly cytosolic, but this does not necessarily imply a predominant role for MT since the cytosolic fraction also includes other macromolecules which may be the target binding site for accumulated trace metals.     

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary,in and out of the reproductive season

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.     

Evaluation of cadmium,lead and metallothionein contents in the tissues of mussels (Mytilus galloprovincialis) from the Campania coast (Italy): Levels and seasonal trends

The biological effect of seasonality on cadmium, lead and metallothionein contents was assessed in mussels Mytilus galloprovincialis from natural banks located along the coastline of the Gulf of Naples (Campania, Italy). Heavy metals and metallothionein concentrations were measured in digestive and reproductive glands. The results showed a clear correlation between metallothionein content and the reproductive gland status determined during the seasons; on the contrary, no correlation was found between metallothionein and metal contents. Data allow us to hypothesize that metallothionein functions go beyond metal detoxification, thus opening new scenarios for these proteins in invertebrates. The effect of seasons on metals concentration in mussel tissues showed similar seasonal patterns between the sites, regardless of their anthropogenic impacts. Cadmium content was not strictly related to seasonal periods, whereas lead content was significantly lower in summer. The results also indicate that the metal contents in mussels from the Gulf of Naples do not represent a risk to human health, even in the period of their maximum accumulation, and that the relaying of mussels before marketing could improve the animal stress conditions, but having a slight effect on metal excretion.     

Quantitative competitive (qc) RT-PCR as a tool in biomarker analysis

All inducible proteins which respond to known pollutants are potential molecular biomarkers. Quantitative competitive (qc) RT-PCR represents a uniquely sensitive tool for measuring the extent of induction of molecular biomarkers such as metallothionein, which is responsive to inducers that include a range of heavy metals. Using the yellowbelly flounder Rhombosolea leporina collected from sites in the Manukau Harbour and Hauraki Gulf (Auckland, New Zealand) as an indicator species, we describe the methodology underpinning the use of qcRT-PCR as a tool in biomarker analysis with reference to the induction of metallothionein. The results show reasonable correlation between the extent of metallothionein induction and the liver burden of Cu and Zn.     

Concentrations and distribution of Fe,Zn and Cu in tissues of the white sucker (Catostomus commersoni) in relation to elevated levels of metals and low pH

This study examines whether the process of lake acidification influences the accumulation of Fe, Zn and Cu in the tissues of the white sucker (Catostomus commersoni). Concentrations of Fe, Zn and Cu were measured in the liver, kidney and muscle of white sucker sampled from 4 acidic (pH range 4.8–5.3), 1 slightly acidic (pH = 5.8) and 3 circumneutral (pH = 6.3, 6.4) lakes located in south-central Ontario, Canada. Pearson product-moment correlation coefficients were used to determine relationships between average elemental concentrations in the 3 tissues and both sediment and water metal concentrations plus lake pH, DOC and alkalinity. Despite the 1000-fold difference in H+ concentration among the 8 study lakes, tissue concentrations of Fe, Zn and Cu did not correlate with lake pH. Average Fe, Zn and Cu tissue concentrations did not correlate with metal concentrations in lake water. Only Zn concentrations in the liver and muscle were correlated with Zn concentrations in the sediment (r = 0.83 and r = 0.88, P < 0.05). Iron and Cu were regulated by the white sucker over a wide range of lake pH and metal concentrations in both the water and sediment. In contrast, Zn tissue concentrations were correlated with sediment Zn concentrations, the latter are thought to result from Zn inputs of anthropogenic origin.     

Molecular defense systems are expressed in the king bolete (Boletus edulis) growing near metal smelters

The induction of defense systems against metal exposure was investigated in 48 wild-growing fruiting bodies of the king bolete (Boletus edulis) from two areas polluted with several transition metals from smelters, as well as five reference areas. To determine the degree of metal exposure, cadmium (Cd), zinc (Zn), and copper (Cu) were determined in caps of fruiting bodies by atomic absorption spectrophotometry (AAS), whereas mercury (Hg) was determined by cold vapor atomic fluorescence spectrometry (CVAFS). Caps were analyzed further with respect to relative activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as concentrations of total glutathione (GSHTOT = GSH + GSSG) and relative concentrations of heat shock protein 70 kDa (HSP70). The results showed that concentrations of the four metals, as well as SOD, CAT and HSP70, were significantly elevated in the exposed group (Mann-Whitney, P < or = 0.001). In contrast, GSHTOT was significantly lowered in the exposed group (P < or = 0.05). Significant positive correlations were established between concentrations of Cd, Zn, Hg, or Cu and activities of SOD (Spearman's P < or = 0.01 for the association between SOD and Cd, P < or = 0.001 for all other metal exposure parameters), CAT (P < or = 0.001 for all exposure parameters), or expression of HSP70 (P < or = 0.001 for all exposure parameters). Significant negative correlations were found between total GSH and Cd (P < or = 0.001), Zn (P < or = 0.001), or Hg (P < or = 0.05). We conclude that antioxidant enzymes are induced in wild-growing B. edulis exposed to environmentally relevant concentrations of potentially toxic transition metals; whereas the net consumption of GSH that occurs with increasing metal exposure may reflect GSH consumption by mechanisms of metal detoxification. Finally, the induction of HSP70 suggests that the antioxidant response and the mechanisms in which GSH is consumed are insufficient for protection against the harmful effects of severe metal stress.     

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY–COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM‐coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long‐term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long‐term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.     

Metal Tolerance and Accumulation Ability of the Ni Hyperaccumulator Streptanthus polygaloides Gray (Brassicaceae)

High concentrations of metals occur in some plant species (termed hyperaccumulators), such as the Ni hyperaccumulator Streptanthus polygaloides. We determined the tolerance of S. polygaloides to, and its accumulation abilities for, six metals (Ni, Zn, Cu, Co, Mn, and Pb). Potting mix concentrations used for all metals ranged from 0 to 1200 μg/g dry weight. For Ni, a treatment of 1600 μg/g was included. For Mn, treatments of 1600, 2000, and 2500 μg/g also were used, and for Pb these concentrations plus 3500 μg/g were included. Germination, plant number per pot, and size at days 30 and 39, number of plants at the end of the experiment (day 49), flower production, and metal concentration in the aboveground biomass were documented. Lead and Ni showed no consistent effects on plant performance, but yielded increased tissue metal concentrations. Streptanthus polygaloides was more sensitive to Co, Cu, and Zn, as ≥ 400 mg/g significantly suppressed plant growth, survival, and flower production. Tissue metal concentrations also were increased to maxima of 1500 μg Co/g, 120 μg Cu/g, and 6000 μg Zn/g. Manganese affected S. polygaloides less markedly, as ≥ 800 mg/kg decreased growth, survival, and flower production. Maximum tissue Mn concentration was 2900 μg/g. We concluded that S. polygaloides would be an appropriate phytoextractor for soils contaminated with Ni or low levels of Co but would not be useful for Cu, Zn, Mn, and Pb.     

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd,Cu and Pb exposure

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.     

Stress responses and metal tolerance of <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> in metal-enriched lake water and artificial medium

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.     

Metal concentrations and metallothionein metal detoxification in blue sharks,Prionace glauca L. from the Western North Atlantic Ocean

BackgroundElasmobranchs are particularly vulnerable to environmental metal contamination, accumulating these contaminants at high rates and excreting them slowly. The blue shark Prionace glauca L. is one of the most heavily fished elasmobranchs, although information regarding metal contamination and detoxification in this species is notably lacking.MethodsBlue sharks were sampled in the western North Atlantic Ocean, in offshore waters adjacent to Cape Cod, Massachusetts. Total and metallothionein-bound liver and muscle metal concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS), metallothionein detoxification and oxidative stress endpoints were determined by UV–vis spectrophotometry.ResultsMetallothionein detoxification occurred for As, Cd, Cs, Cu, Hg, Pb, Se, Ti and Zn in liver, and for As, Cd, Cs, Pb, Se, and Zn in muscle, while reduced glutathione defenses seem to be related to Co and Zn exposure.ConclusionThis is the first report for several metals (Ag, Co, non-radioactive Cs, Sb, Ti and V) for this species, which will aid in establishing baseline elemental data for biomonitoring efforts, health metrics, and conservation measures.     

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.     

In Vitro Radiation Induced Alterations in Heavy Metals and Metallothionein content in Plantago ovata Forsk

Proton Induced X-ray emission (PIXE) and fluorescence-activated cell sorting (FACS) have been used to study the effects of gamma irradiation on heavy metal accumulation in callus tissue of Plantago ovata-an important cash crop of India. PIXE analysis revealed radiation-induced alteration in trace element profile during developmental stages of the callus of P. ovata. Subsequent experiments showed antagonism between Fe and Cu and also Cu and Zn and synergistic effect between Fe and Zn. FACS analysis showed significant induction of the metallothionein (MT) protein following gamma-irradiation, and maximum induction was noted at the 50-Gy absorbed dose. This indicated a progressive increment of MTs as a measure for protection against gamma-rays, to combat alteration in the homeostasis of heavy metals like Fe, Cu, Zn, and Mn.     

Influence of ultraviolet radiation, copper, and zinc on microcystin content in Microcystis aeruginosa (Cyanobacteria)

Cyanobacterial toxin production is allied to some unknown trigger resulting in the production of toxins such as microcystin. We hypothesize that microcystins serve as metal ligands to control bioavailability and toxicity of ambient metals. Since ultraviolet radiation (UVR) promotes photo-oxidation of organic metal ligands and influences trace metal bioavailability, the present study aimed to investigate the influence of UVR, Cu, and Zn on specific growth rates, biomass, photosynthetic capacity, and microcystin content in Microcystis aeruginosa. Two toxigenic strains of Microcystis were cultivated using either Lake Erie filtered water or a chemically defined medium, with realistic concentrations of Cu and Zn combined with natural or artificial UVR exposure. Cu was more toxic than Zn on the basis of free ion concentration of trace metals in synthetic medium, although in Lake Erie water total added Zn (10 nM) or Zn plus Cu (10 nM) had a more detrimental effect on biomass and specific growth rate. Natural UVR delivered at 25% ambient levels caused no decrease on the parameters measured (chlorophyll-a, photosynthetic rate), yet artificial levels of UVR (up to 5.9 μmol UVB photons m⁻² s⁻¹) negatively affected biomass and specific growth rate. Cellular levels of microcystin (per unit chlorophyll-a) were concomitant with specific growth rather than being triggered in response either of these stressors (UVR, Zn, and Cu) alone or in combination, in agreement with a purported constitutive production of microcystins.     

Metal exposures to native populations of the caddisfly Hydropsyche (Trichoptera: Hydropsychidae) determined from cytosolic and whole body metal concentrations

Metal concentrations of the soluble fraction of the cytoplasm (cytosol) and the whole body were determined in the caddisfly Hydropsyche spp. (Trichoptera). Metal accumulation in the cytosol and the whole body were compared in samples collected along 380 kms of a contamination gradient in the Clark Fork river in four consecutive years (1992–1995), and from a contaminated tributary (Flint Creek). Samples from the contaminated sites were compared to an uncontaminated tributary (Blackfoot River). Relations between cytosolic metal concentration and cytosolic protein (used as a general biomarker of protein metabolism) also were examined in 1994 and 1995. Relative to whole body concentrations, cytosolic metal concentrations varied among metals and years. Spatial patterns in whole body and cytosolic Cd, Cu and Pb concentrations were qualitatively similar each year, and these concentrations generally corresponded to contamination levels measured in bed sediments. The proportions of metals recovered in the cytosol of ranged from 12 to 64% for Cd and Cu and from 2 to 38% for Pb. Zinc in the whole body also was consistent with contamination levels, but cytosolic Zn concentrations increased only at the highest whole body Zn concentrations. As a result, the proportion of Zn recovered in the cytosol ranged from 16 to 63% and tended to be inversely related to whole body Zn concentrations. The proportions of cytosolic metals varied significantly among years and, as a result, interannual differences in metal concentrations were greater in the cytosol than in the whole body. The results demonstrated that Hydropsyche in the river were chronically exposed to biologically available metals. Some features of this exposure were not evident from whole body concentrations. In general, protein levels did not correspond to cytosolic metal concentrations. A variety of environmental factors could interact with metal exposures to produce complex responses in protein metabolism. Systematic study will be necessary to differentiate the effects of multiple environmental stressors on organisms living in contaminated ecosystems. This revised version was published online in July 2006 with corrections to the Cover Date.     

METAL‐INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation‐sensitive reporter dye, 5‐(and‐6)‐carboxy‐2′,7′‐dihydrodifluorofluorescein diacetate (H₂DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10^?6 to 10^?9 M, except in the case of Fe(III), which was effective at 10^?18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.