Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R² = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.     

The dichotomy of mechanisms of copper accumulation in yeast induced by enhanced levels of copper as well as menadione in growth media

Abstract

A high copper accumulation is induced in the yeast Saccharomyces cerevisiae either by menadione at the level of 200 μM in the growth medium, or by elevated concentrations of copper. While the uptake, as well as the toxicity of copper, strongly depend on the zinc concentration in the medium, there is no influence of zinc on copper intake induced by menadione. The copper binding ligand d-penicillamine suppresses the accumulation of copper in the menadione systen, whereas it has virtually no effect in the copper system. Within the range of non-toxic concentrations, copper is predominantly taken up by an energy-dependent mechanism. In contrast, the accumulation in the menadione system is clearly energy-independent. Thus there exist at least two different mechanisms for the uptake of copper in the yeast Saccharomyces cerevisiae.     

COMPARATIVE pH DEPENDENT METAL INHIBITION OF NUTRIENT UPTAKE BY SCENEDESMUS QUADRICAUDA(CHLOROPHYCEAE)1,2

Cadmium and copper inhibition of nutrient uptake by the green alga Scenedesmus quadricauda is highly pH dependent in an inorganic medium; both metals are less toxic at low pH. The alga was grown in chemostats with both N and P approaching limiting levels; it was then possible to study metal toxicity to the nitrate, ammonium, and phosphate uptake systems of algae in an identical physiological state. When the logarithm of the Cd concentration causing 25% inhibition of nitrate, ammonium, and phosphate uptake was regressed against pH almost perfect linear relationships were obtained. This was also true at the 50% inhibition level, except for a smaller than predicted increase in Cd toxicity to ammonium uptake at pH 8, which may be due to the beginning of Cd precipitation at this pH. Cu²⁺ toxicity was linearly related to pH for ammonium and phosphate uptake and although, its toxicity for nitrate uptake also increased with pH, the increase was not perfectly linear. The toxicity of total Cu showed no linear relationship to pH. Cd²⁺ and Cu²⁺ toxicity increased by up to four orders of magnitude from pH 5 to 8. Competition between free metal and hydrogen ions for uptake sites on the cell surface is suggested as a mechanism increasing the toxicity of free metal, ions as the hydrogen ion content decreases (i.e. at higher pH).     

Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it's attenuation by zinc: ex vivo

Copper and zinc act as a cofactor of over 300 mammalian proteins. Both have same electronic configuration therefore they are antagonist at higher individual concentration. The present study was designed with the aim to investigate the mechanisms pertaining to toxic effects of copper on human peripheral blood mononuclear cells (PBMCs) and to evaluate the cytoprotective effect of zinc on copper-induced cytotoxicity. The copper uptake into PBMCs was progressively increased with increasing concentration of metal in the growth medium. However, no significant effect on copper uptake was observed in the presence of zinc. Cell proliferation rate was decreased with increasing copper concentration. Interestingly, the proliferation rate of zinc treated PBMCs remained nearly the same as that of control cells. LD₅₀ of copper (115 μM) was increased six times (710 μM) in presence of zinc for PBMCs. At higher concentrations of copper (> 100 μM) decrease level of GSH was noticed. Increased levels of metallothionein in PBMCs were observed in response to zinc. DNA fragmentation studies also showed that copper produced DNA fragmentation at LD₅₀ (115 μM). Subsequently, zinc showed protection against DNA fragmentation caused by copper. Cell structure of PBMCs at LD₅₀ (115 μM copper) showed membrane bound cystic spaces and mitochondria having disrupted cristae and few myelin figures. In presence of zinc at LD₅₀ of copper (115 μM) cells showed improvement in mitochondrial structure and membrane bound cystic spaces. Taken together, the results of our study demonstrates that zinc play an important role in prevention of copper toxicity in peripheral blood mononuclear cells.     

Phylogenetic differences in cardiac activity, metal accumulation and mortality of limpets exposed to copper: A prosobranch-pulmonate comparison

Taxonomic relationships for pollutant tolerance in marine invertebrates are surprisingly poorly known, despite being potentially useful for pollution biomonitoring. A popular view is that cellular and molecular adaptations for natural stress may be important in tolerating pollution. We compared the physiological and mortality responses to copper (Cu²⁺) of limpets from two different lineages: the Prosobranchia (Patellogastropoda: Helcion concolor and Cellana capensis) and the Pulmonata (Siphonaria serrata and Siphonaria capensis). Copper tolerance was apparently more closely related to phylogenetically-based physiology, than to tolerance of desiccation and or heat. The Siphonaria limpets were nearly an order of magnitude more tolerant of copper than the patellogastropod limpets, even though S. serrata has the lowest intertidal distribution. Initial copper exposure (0.25 ppm Cu²⁺ for 2 h) induced heart rate depression in Siphonaria (to around 50% of the baseline rate), while their tissue copper concentrations remained at the relatively high control levels. Copper exposure (0.25 ppm Cu²⁺ for 2 h) had no effect on heart rate of the patellogastropod limpets, but led to a significant increase in tissue copper. These results suggest that enhanced copper tolerance by Siphonaria relates to cardiac depression and a concomitant metabolic depression. Such physiological attributes are implicated in prolonged behavioural isolation, involving pneumostome closure and shell clamping, which is likely to reduce the uptake of copper. Furthermore, better regulation of internal copper levels by Siphonaria, is suggested by their exclusive possession of blood haemocyanin. Dependence on relatively high aerobic metabolism by the patellogastropod limpets, would limit their capacity for isolation and pollutant avoidance.     

Cadmium and zinc uptake,growth, and primary production inCoscinodiscus granii cultures containing low levels of cells and dissolved organic carbon

Coscinodiscus granii Gough was cultivated at low cell densities in aged Atlantic sea water containing very little dissolved organic carbon; the water was enriched with low levels of nutrients but no chelators were added. Cadmium additions provided final concentrations of 0.1 to 26.5 μg Cd l⁻¹, zinc being kept constant at a level of 38 μg Zn l⁻¹. Carrier-free¹⁰⁹Cd and⁶⁵Zn were used as tracers for the two metals. Growth in terms of cell numbers and primary productivity capacity, using the¹⁴C uptake rate, was followed during the exponential growth phase for the first 5 days of the experiment and then for a further 3 days during the stationary phase. On each day, the metal contents of the cells were determined. Cadmium concentrations of 20 μg Cd l⁻¹ and more resulted in growth inhibition whereas 17.5 μg Cd l⁻¹ reduced the growth only slightly. The physiological state of the cells influenced the heavy-metal uptake per cell at sublethal Cd levels. Dead cells had a higher heavy-metal concentration than living cells. Microscopical observations revealed that cells just about to divide were less sensitive to a given toxic heavy-metal concentration than cells which had recently divided. This might have been due to different surface/volume ratios.     

Physiological responses of wheat genotypes grown in chelator-buffered nutrient solutions with increasing concentrations of excess HEDTA

The chelator-buffered nutrient solutions containing excess chelator have been used frequently in the micronutrient research, but potential toxicity of the excess chelator has not been ascertained. The present study was conducted to test effects of four concentrations of excess HEDTA [ N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid] and two levels of total Zn on growth, root exudation, and nutrient uptake and transport by Triticum aestivum L. (cv. Aroona) and Triticum turgidum L. conv. durum (Desf.) MacKey (cv. Durati) genotypes differing in tolerance to Zn deficiency. Excess HEDTA at 50 μM reduced root and shoot growth and caused visual toxicity symptoms (necrotic lesions) on leaves; these effects were generally absent at lower concentrations of excess HEDTA. Root exudation of phytosiderophores increased with increasing concentrations of excess HEDTA at deficient and sufficient Zn levels, and was higher in Zn-deficiency-tolerant Aroona than in Zn-deficiency-sensitive Durati wheat. Shoot and root Zn concentrations showed a saturable response to increasing Zn²⁺ activities in solution. Excess HEDTA at 50 μM caused an increase in shoot concentrations of Fe and a decrease in concentrations of Mn and Cu. An average rate of Zn uptake increased with an increase in Zn²⁺ ionic activity in solution, with Zn-deficiency-tolerant Aroona having a higher rate of Zn uptake than Zn-deficiency-sensitive Durati in the deficiency range of Zn²⁺ activities. Average uptake rates of Mn and Cu decreased with an increase in concentration of excess HEDTA. Similar observations were noted for transport of Mn and Cu to shoots, while Zn transport to shoots was proportional to Zn²⁺ activities in solution. It was concluded that excess HEDTA at 50 μM adversely affects wheat growth and physiology, while excess of 25 μM or less does not cause measurable toxicity. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of contamination of soil with copper,lead and arsenic on the growth and composition of plants

Summary Accumulations of copper, lead and arsenic in soils affected by orchard sprays or mining were investigated in relation to their effects on growth and composition of plants. Seasonal variations in concentrations of the elements in pasture plants sampled from contaminated soils in the field are reported. The effects of soil temperature and applications of the nutrients P, S and N on the composition of plants grown in contaminated soils were investigated in glasshouse experiments.The copper concentrations of pasture species sampled from sites which were formerly orchards were usually high (20 to 60 mg kg^–1) during most of the growing season and may present some risk of toxicity to grazing ruminant animals. Lead (0.8 to 21 mg kg^–1) and arsenic (<0.2 to 5.8 mg kg^–1) concentrations were within, or close to the normal range of concentration in plants. In the glasshouse experiments, soil temperature was found to be an important factor in the uptake of copper, lead and arsenic. There were significant differences in uptake between genotypes. Applications of fertilizers at rates equivalent to those used for commercial vegetable production generally resulted in small decreases in the concentrations of copper, lead and arsenic concentrations in silver beet.     

COPPER TOXICITY TO SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

Copper toxicity to Skeletonema costatum (Grev.) Cleve has been studied in batch cultures of chemically defined culture media. The alga is relatively insensitive to cupric ion activity, demonstrating no effect on growth up to (Cu²⁺) = 10^?8.5 M. Cultures inoculated from stationary phase stocks exhibit a prolongation of the lag phase with increasing copper concentrations near and above the point of precipitation of the copper. The toxicity of copper is a function of the silicic acid concentration in the medium. This effect is observed in a range of Si(OH)₄ concentrations (10^?5 M to 10^?4 M) above known values for the saturation of silicon uptake kinetics, thus suggesting an influence of copper on silicate metabolism.     

Growing,physiological responses and Cd uptake of Corn (Zea mays L.) under different Cd supply

The effects of Cd on the growth and Cd uptaking in corn (Zea mays L.) were explored under different Cd stress. The results showed that no reduction in shoot and root dry matter yields were noted when the plants were grown at Cd supply levels ≤100 μmol l^?1 nutrient solution. The Cd concentration in the shoots and roots of corn increased sharply with increasing external Cd supply levels, peaked at 50 μmol l^?1, and then decreased slowly with further increasing Cd levels due to high Cd toxic effects on root growth. The concentrations of chlorophyll a, chlorophyll b and chlorophyll a + b in the leaf of corn decreased slowly with increasing external Cd supply levels. Proline concentrations of corn increased when the plants were grown under the external Cd influence. The lower concentration of Cd treatments did not influenced the growth of corn significantly, and increased the uptake of Cd, the higher levels of Cd supply caused significantly physiological resposes and decreased the Cd uptaking.     

Chromium removal from water by three species of dyckweeds

The effects of exogenous chromium (Cr) on the growth of three species of duckweeds: Spirodela punctata (G.F.W. Meyer) Thomps., Spirodela polyrhiza (L.) Schleiden and Lem na gibba L., were examined at Cr concentrations ranging from 0.1 to 20 p.p.m. Using ⁵¹Cr radiotracer methodology, it was demonstrated in Experiment 1 that sorption was directly related to ambient Cr concentrations after 8 days of exposure. At and above 1.0 p.p.m. Cr, negative effects on growth were evident, with seasonal variations apparently also affecting Cr uptake and toxicity. Chrome tolerance, based on the percentage of control growth achieved by exposed plants, showed S. polyrhiza to be the least tolerant to Cr, while S. punctata and L. gibba were more tolerant at concentrations above 1.0 p.p.m. In Experiment 2, duckweeds grown in nutrient-rich water with an initial concentration of 10 p.p.m. exhibited substantial growth, with bioaccumulation of Cr increasing with time.The possible use of various types of biological treatment systems for the removal of Cr from wastewater is discussed.     

Effects of NH4+ concentration on growth,morphology and NH4+ uptake kinetics of Salvinia natans

Many plants develop toxicity symptoms and have reduced growth rates when supplied with ammonium (NH₄⁺) as the only source of inorganic nitrogen. In the present study, the growth, morphology, NH₄⁺ uptake kinetics and mineral concentrations in the tissues of the free-floating aquatic plant Salvinia natans (water fern) supplied exclusively with NH₄⁺–N at concentrations of 0.25–15 mM were investigated. S. natans grew well, with relative growth rates of c. 0.25 g g^?1 d^?1 at external NH₄⁺ concentrations up to 5 mM, but at higher levels growth was suppressed and the plants had small leaves and short roots with stunted growth. The high-affinity transport system (HATS) that mediate NH₄⁺ uptake at dilute NH₄⁺ levels was downregulated at high NH₄⁺ concentrations with lower velocities of maximum uptake (V_max) and higher half-saturation constants (K_1/2). High NH₄⁺ levels also barely affected the concentrations of mineral cations and anions in the plant tissue. It is concluded that S. natans can be characterized as NH₄⁺-tolerant in line with a number of other species of wetland plants as growth was unaffected at NH₄⁺ concentrations as high as 5 mM and as symptoms of toxicity at higher concentrations were relatively mild. Depolarization of the plasma membrane to the equilibrium potential for NH₄⁺ at high external concentrations may be a mechanism used by the plant to avoid excessive futile transmembrane cycling. S. natans is tolerant to the high NH₄⁺ levels that prevail in domestic and agricultural wastewaters, and the inherent high growth rate and the ease of biomass harvesting make S. natans a primary candidate for use in constructed wetland systems for the treatment of various types of nitrogen-rich wastewaters.     

Sublethal Toxicity Endpoints of Heavy Metals to the Nematode Caenorhabditis elegans

Caenorhabditis elegans, a free-living nematode, is commonly used as a model organism in ecotoxicological studies. The current literatures have provided useful insight into the relative sensitivity of several endpoints, but few direct comparisons of multiple endpoints under a common set of experimental conditions. The objective of this study was to determine appropriate sublethal endpoints to develop an ecotoxicity screening and monitoring system. C. elegans was applied to explore the sublethal toxicity of four heavy metals (copper, zinc, cadmium and chromium). Two physiological endpoints (growth and reproduction), three behavioral endpoints (head thrash frequency, body bend frequency and feeding) and two enzymatic endpoints (acetylcholine esterase [AChE] and superoxide dismutase [SOD]) were selected for the assessment of heavy metal toxicity. The squared correlation coefficients (R²) between the responses observed and fitted by Logit function were higher than 0.90 and the RMSE were lower than 0.10, indicating a good significance statistically. There was no significant difference among the half effect concentration (EC50) endpoints in physiological and behavioral effects of the four heavy metals, indicating similar sensitivity of physiological and behavioral effects. AChE enzyme was more sensitive to copper, zinc, and cadmium than to other physiological and behavioral effects, and SOD enzyme was most sensitive to chromium. The EC50 of copper, zinc, and cadmium, to the AChE enzyme in the nematodes were 0.68 mg/L, 2.76 mg/L, and 0.92 mg/L respectively and the EC50 of chromium to the SOD enzyme in the nematode was 1.58 mg/L. The results of this study showed that there was a good concentration-response relationship between all four heavy metals and the sublethal toxicity effects to C. elegans. Considering these sublethal endpoints in terms of simplicity, accuracy, repeatability and costs of the experiments, feeding is the relatively ideal sublethal toxicity endpoint of heavy metals to C. elegans.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.     

Increase of Copper Toxicity to Growth of Chlorella vulgaris with Increase of Light Intensity

Abstract In the absence of inhibitory concentrations of copper, the photoautotrophic growth of Chlorella vulgaris INETI58C at 27°C exhibited a higher specific growth rate and reached a higher maximal concentration of biomass, under irradiance of 150 W m⁻², compared with 100 W m⁻². However, when the mineral growth medium was supplemented with CuSO₄ (range 40–80 μM), algal growth was significantly affected at the higher light intensity. In the presence of Cu²⁺, the increase in dry biomass was uncoupled from the increase in cell concentration since more than 16 autospores gathered together, inside the enlarged mother cell, suggesting that copper arrested the normal bursting of the mother cell wall. At the higher irradiance, growth medium supplementation with 80 μM of CuSO₄ led to bleaching of photosynthetic pigments. No growth was observed, while, under the lower irradiance, growth was only slightly inhibited. Results clearly showed that copper toxicity to growth of C. vulgaris was strongly influenced by light intensity. Higher light intensity elicits lethal or sublethal Cu²⁺ damage at concentrations lower than the threshold level for damage at lower light intensities. Cu²⁺ may elicit lethal or sublethal light damage at irradiances lower than the threshold level for unpolluted aquatic systems. Received: 17 January 1997; Accepted 15 April 1997     

Effects of heavy metals on Tetraselmis suecica: Ultrastructural and energy-dispersive X-ray spectroscopic studies

The influence of metal contamination on the marine alga Tetraselmis suecica was investigated at physiological and ultrastructural levels. For this analysis, the growth response of this microalga was studied after the addition of various concentrations of heavy metals (Cd, Cu). The concentration corresponding to 50% growth inhibition (IC₅₀) and the number of days per cell cycle (Td) studied, revealed that the toxic effects of copper are heavier than those of cadmium. In the case of copper contamination, the Td grows with increasing metal concentration in the culture medium, while it remains unchanged during the cadmium contamination. The toxicity of cadmium, only observed in the latency phase of growth, suggests an adaptation phenomenon of T suecica to this metal. Ultrastructural changes in response to pollutants were investigated; copper induced cytoplasmic vacuolisation, organelle changes, appearance of cells with multilayered cell walls and excretion of organic matter. In the case of cadmium contamination, ultrastructural changes mainly affected the osmiophilic vesicles, of which both number and volume increased with increasing metal concentration in the culture medium. The results of X-ray microanalysis revealed that Cd and Cu were strongly present in excreted organic matter and osmiophilic vesicles. The latter can be excreted during cell division, thus participating in detoxification processes. Intracellular cadmium incorporation proved that some toxic effects of this metal are a result of interaction with endogenous cellular constituents. In the case of copper contamination, the presence of copper in walls of a multilayered cell suggests that these structures constitute an additionnal adsorbing area for this element, reducing metal free concentration in the medium. Mechanisms of metal detoxification of Tetraselmis suecica are discussed.     

Effects of high copper concentrations on soil invertebrates (earthworms and oribatid mites):

Data in the literature on the toxicity and uptake of copper by soil invertebrates are contradictory. Copper toxicity and bioaccumulation studies were therefore performed using earthworms and oribatid mites. Field-simulating experiments in soil-filled plastic containers showed that earthworms try to escape moderately toxic situations and that they are much more sensitive than oribatid mites to temporary high Cu²⁺ concentrations in soils. The total copper concentration in the bodies of the earthworm species Octolasium cyaneum was measured in experiments with different soil types and different amounts of added CuSO₄. The copper concentrations in the earthworms increased in response to the higher concentrations of the copper fraction extractable with 2.5% acetic acid in the soil. Furthermore, internal copper concentrations showed a slight tendency to oscillate. The worms died when the concentrations within their bodies exceeded about 100–120 ppm, calculated on a dry weight basis. To interpret the experimental results, a compartment model is proposed which describes the dynamics of different fractions of copper in worms living in varying soil environments. Applying this model, the different reports on toxicity and uptake of copper in the literature no longer contradict each other.     

The mobilisation and fate of soil and rock phosphate in the rhizosphere of ectomycorrhizal Pinus radiata seedlings in an Allophanic soil

A pot experiment was conducted to investigate the uptake of Zn from experimentally contaminated calcareous soil of low nutrient status by maize inoculated with the arbuscular mycorrhizal (AM) fungus Glomus caledonium. EDTA was applied to the soil to mobilize Zn and thus maximize plant Zn uptake. The highest plant dry matter (DM) yields were obtained with a moderate Zn addition level of 300 mg kg^?1. Plant growth was enhanced by mycorrhizal colonization when no Zn was added and under the highest Zn addition level of 600 mg kg^?1, while application of EDTA to the soil generally inhibited plant growth. EDTA application also increased plant Zn concentration, and Zn accumulation in the roots increased with increasing EDTA addition level. The effects of inoculation with G. caledonium on plant Zn uptake varied with Zn addition level. When no Zn was added, Zn translocation from roots to shoots was enhanced by mycorrhizal colonization. In contrast, when Zn was added to the soil, mycorrhizal colonization resulted in lower shoot Zn concentrations in mycorrhizal plants. The P nutrition of the maize was greatly affected by AM inoculation, with mycorrhizal plants showing higher P concentrations and P uptake. The results indicate that application of EDTA mobilized soil Zn, leading to increased Zn accumulation by the roots and subsequent plant toxicity and growth inhibition. Mycorrhizal colonization alleviated both Zn deficiency and Zn contamination, and also increased host plant growth by influencing mineral nutrition. However, neither EDTA application nor arbuscular mycorrhiza stimulated Zn translocation from roots to shoots or metal phytoextraction under the experimental conditions. The results are discussed in relation to the environmental risk associated with chelate-enhanced phytoextraction and the potential role of arbuscular mycorrhiza in soil remediation.     

Cadmium-induced changes in growth and cell cycle gene expression in suspension-culture cells of soybean

The toxic effects of cadmium on growth and development of living organisms are well documented. However, the molecular mechanisms responsible for the inhibition of plant growth by cadmium are still not completely understood. We determined the effects of cadmium concentrations in the range of 1–11 μM on the growth of Glycine max L. cv. Navico suspension-culture cells, as well as on the expression of two cell cycle genes: cyclin B1 and cyclin-dependent type A kinase (CDK-A). There was no detectable decrease in cell viability at any tested Cd²⁺ concentrations. The lower concentrations of Cd²⁺ (1–4 μM) stimulated cell culture growth; however, this did not correspond with increased expression of cell cycle genes. The inhibition of cell growth was observed at concentration of Cd²⁺ higher than 6 μM. Interestingly, it correlated well with the decreased cyclin B1 mRNA levels, but had no significant effect on the levels of CDK-A mRNA.     

Copper toxicity to five Parmelia lichens in vitro

Treatment of Parmelia caperata, P. perlata, P. subrudecta, P. sulcata and P. tiliacea with CuSO₄ resulted in a time- and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus, indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached intracellular sites in the thallus, and K⁺/Cu²⁺ exchange was not electroneutral. After 5 h of exposure to copper, the extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with copper absorption levels, but only at 0.05<P<0.10, suggesting that membrane damage was proportional to the amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall. Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic, and usnic acids. Titration of the extracts with copper and assay of the free Cu²⁺ concentration revealed the presence of copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu²⁺ to each of the lichen substances detected in the extracts. However, no significant correlation (P>0.10) was found between the Cu²⁺-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a simple function of the Cu²⁺-binding properties of the lichen substances present in the thallus. Several hypotheses were formulated to interpret the results.