Molecular fractionation of zn,cu, cd and pb in the midgut gland of Helix pomatia l.

1. Specimens of Helix pomatia were fed lettuce enriched with Zn, Cu, Cd and Pb for 30–35 days, after which time the distribution of the four metals in the midgut gland was studied by centrifugation, ion exchange and gel chromatography.2. In control snails the distribution between the soluble and particulate fraction of the midgut gland is relatively uniform, with a ratio of approximately 3:1 for Cu, Cd and Pb, and of 1:1 for Zn. However, after metal-loading about two-thirds of total Zn and Pb appears in the particulate fraction whereas little change in relative distribution is observed with Cu and Cd.3. After ion exchange chromatography of the supernatant both Cd and Zn eluted in one distinct peak, Cu in two peaks, whereas Pb seemed to be distributed unspecifically throughout the elution volume.4. If ion exchange is followed by gel chromatography, Zn, Cu and Cd are found associated with from one to four molecular species, ranging in mol. wt from 1000 to > 20,000, whereas no complex was found for Pb. The molecular patterns are specific for each of the three metals and there are some specific shifts in abundance, usually involving one molecular fraction, if control snails are compared with metal-loaded snails.     

Nematode and snail metallothioneins

Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.     

Cadmium-bound metallothionein induces apoptosis in rat kidneys, but not in cultured kidney LLC-PK1 cells

The ability of cadmium-bound metallothionein(Cd-MT) to induce apoptosis was investigated in vivo and in vitro. Administration of purified Cd-MT (0.15 mg MT bound Cd per kg body weight) to the rat induces DNA fragmentation, a biochemical characteristic of apoptosis in the kidney at 16 h, which was detectable by ethidium bromide staining on an agarose gel. It was still detected 24 h after administration. Induction of apoptosis by Cd-MT was specific to kidney; it was not observed in cerebrum, cerebellum, heart, lung, liver, testis, dorsolateral prostate, and ventral prostate. In contrast, addition of Cd-MT (0.01-100 microM) to the cultured porcine kidney LLC-PK1 cells failed to induce apoptosis under the condition where cadmium chloride (10 microM) did. There was no additivity of induction of apoptosis by CdCl2 (10 microM) in the presence of Cd-MT (0.01-100 microM). To examine the effect of intracellular MT on cadmium-induced apoptosis in cultured cells, new cell lines were established, which constitutively produce MT, being termed as Cd(r)-LLC-PK1 cells since Cd-MT exogenously added had much less permeability to the cultured cells. Followed by exposure of wild-type LLC-PK1 cells to 50 microM CdCl2 for 24 h, the surviving cells(Cd(r)-LLC-PK1 cells) induce MT at the level of 1.9 microg/2 x 10(6) cells. In Cd(r)-LLC-PK1 cells, 10 microM CdCl2 failed to induce apoptosis, but 60 microM CdCl2 could exert the apoptotic response, indicating that intracellular MT which was induced by CdCl2 did not facilitate CdCl2-elicited apoptosis. Furthermore, chromatin in rat kidneys was condensed by Cd-MT, but not that in LLC-PK1 cells. Thus, Cd-MT induces apoptosis in rat kidneys, but not in the cultured renal cells, suggesting that the ionic form of cadmium was required for programmed cell death.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Patterns of accumulation,distribution and liberation of zn,cu, cd and pb in different organs of the land snail Helix pomatia L

1. The snail Helix pomatia was fed lettuce enriched with zinc, cadmium, lead and copper for 32 days, followed by a feeding period of 40–50 days with uncontaminated lettuce. The time-dependent distribution of the four metals during loading and unloading was determined in all major organs. Four general types of response to metal loading have been distinguished. Another general feature is the rhythmic nature of accumulation and de-accumulation of metals in several organs. On the other hand, each metal also elicits specific responses which can be described in terms of acute or delayed reaction and long-term accumulation.2. Lead is accumulated by most organs and redistributed after loading has stopped, but a single organ, the midgut gland, accumulates about 90% of this metal.3. Zinc accumulates in most organs during the first part of the loading period and is then redistributed to the midgut gland which may hold up to 70% of the metal at the end of the experiment.4. Cadmium is not taken up at all by a number of organs but the midgut gland and anterior gut keep accumulating it. After loading has been terminated the metal appears to move from the midgut gland to the gut.5. Copper is distributed more evenly than the other metals in the organs of the snail, the midgut gland not playing a dominant role in the storage of this metal.     

Cadmium uptake in rat hepatocytes in relation to speciation and to complexation with metallothionein and albumin

Cadmium (Cd) uptake has been studied in primary cultures of rat hepatocytes focusing on the impact of inorganic and organic speciation. Uptake time-course studies over a 60-min exposure to 0.3 microM (109)Cd revealed a zero-time uptake and a slower process of accumulation which proceeds within minutes. (109)Cd uptake showed saturation kinetics (K(m) = 3.5 +/- 0.8 microM), and was highly sensitive to inhibition by Zn and Hg. There was no evidence for sensitivity to the external pH nor for any preferential transport of the free cation Cd(2+) over CdCl(n) (2-n) chloro-complexes. According to the assumption that only inorganic metal species are available, metal uptake decreased upon albumin (BSA) addition to the exposure media. In contrast, higher levels of (109)Cd accumulation were obtained under optimal conditions for Cd complexation by MT. Comparison among uptake data obtained under inorganic and organic conditions revealed that Cd-MT would be taken up 0.4 times as rapidly as Cd(inorg). We conclude that uptake of Cd in rat hepatocytes involves specific transport mechanism(s) subjected to Zn or Hg interactions. Uptake of inorganic Cd is not proportional to the levels of free Cd(2+) and does not involve the divalent cation transporter DCT1 nor the co-transporter Fe(2+)-H(+) NRAMP2. We found Cd-MT but not Cd-BSA to be available for the liver cells, and have estimated a binding affinity four orders of magnitude higher for Cd complexation with MT compared to BSA; MT may have a significant role in Cd delivery to the liver.     

Side Effects of Neem Oil on the Midgut Endocrine Cells of the Green Lacewing Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae)

We described the ultrastructure of Ceraeochrysa claveri (Navás) midgut endocrine cells in larva, pupa, and adult, and evaluated the side effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree (Azadirachta indica), on these cells. During the larval period, C. claveri were fed (ad libitum) Diatraea saccharalis (F.) eggs treated with neem oil at concentrations of 0.5%, 1%, or 2%. Transmission electron microscopy showed that two subtypes of endocrine cells, namely granular and vesicular, occurred in the midgut epithelium during the three stages of the life cycle. Both cell types did not reach the midgut lumen and were positioned basally in the epithelium. The endocrine cells did not show extensive infoldings of the basal plasma membrane, and there were numerous secretory granules in the basal region of the cytoplasm. In the granular endocrine cells, the granules were completely filled with a dense matrix. In the vesicular endocrine cells, the main secretory products consisted of haloed vesicles. Ultrastructural examination indicated that only the granular endocrine cells exhibited signs of morphologic changes of cell injury present in all life cycle stages after the larvae were chronically exposed to neem oil by ingestion. The major cellular damage consisted of dilatation and vesiculation of the rough endoplasmic reticulum and the development of smooth endoplasmic reticulum and mitochondrial swelling. Our data suggest that cytotoxic effects on midgut endocrine cells can contribute to a generalized disruption of the physiological processes in this organ due to a general alteration of endocrine function.     

Histopathological effects of larval digenea on the digestive epithelia of the marine prosobranch Cerithidea californica: Fine structural changes in the digestive gland

The effects of natural infections by rediae of the heterophyid Euhaplorchis californiensis on the digestive gland cells of Cerithidea californica (Gastropoda: Prosobranchiata) were studied through the use of light and electron microscopy. Pathological changes in secretory and digestive cells, including the disruption of digestive vacuoles and cell-cell junctions, the deformation of lumenal microvilli, the lysis of distal plasma membranes, and nuclear polymorphism, usually accompany the migration of larval trematodes into the digestive gland. Degenerating secretory cells are further characterized by Golgi complexes which assume an inflated appearance and a reduction of rough endoplasmic reticulum in the cell's perinuclear region. Finally, an increase in excretory and autophagic activities in the digestive gland cells of infected snails may be considered possible mechanisms by which the host is able to compensate for adverse metabolic or nutritional stresses of trematode parasitism.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia.

The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.     

The influence of starvation and copper exposure on the composition of the dorsal carapace and distribution of trace metals in the shore crab Carcinus maenas (L.)

1. The influence of starvation and copper exposure on the composition of the carapace of the shore crab, Carcinus maenas has been investigated. The effects of the concentrations of selected trace metals in the carapace and midgut gland have also been examined.2. Differences in nutritional state did not affect the concentrations or relative proportions of the principle carapace components (calcium, chitin and protein), but starvation was associated with a reduction in carapace copper concentration. Copper concentration in the midgut gland increased in “starved” crabs, while midgut gland zinc and calcium concentrations remained unchanged.3. Starvation in combination with copper exposure (0.5 mg Cu/1) resulted in alterations in both carapace composition and trace metal loads. Carapace calcium concentrations were reduced significantly following copper exposure irrespective of the nutritional state of the crabs. However, the reduction was more marked in “fed” crabs than in “starved” animals.4. Copper-exposed crabs exhibited raised carapace copper concentrations while carapace zinc concentrations were depressed. Such changes were most pronounced in copper-exposed “starved” crabs.5. Calcium concentrations in the midgut glands of “starved” copper-exposed animals were significantly elevated compared with those in either control or “fed”, copper-exposed crabs.6. The findings are discussed with regard to their ecotoxicological significance.     

Advantages and disadvantages of voltammetric method in studying cadmium-metallothionein interactions.

A sensitive and chemical species-selective technique of differential pulse anodic stripping voltammetry (DPASV) was applied in studying the cadmium-metallothionein (Cd-MT) interaction. The amperometric titrations of the purified MT20 and MT10 fractions, isolated by verified biochemical procedures from the digestive gland of cadmium-exposed mussels Mytilus galloprovincialis, with Cd2+ ions were performed in the buffered sodium chloride solution of 0.59 M ionic strength, pH 7.9 and 25 degrees C. Applying the DPASV method at various cadmium to metallothionein ratio several groups of chemical species were recorded. The data on the available ligand concentration to complex cadmium ions (CL), the apparent concentration stability constants (K,) of the respective complexes and the reliability of the determined complexing parameters are discussed. In quantifying the Cd-MT interaction the interference of dithiotreitol (DTT), which is used as the reducing agent in isolation and purification of MTs, is documented.     

Cadmium,chromium, and lead accumulation in aquatic plants and animals near a municipal landfill

This research was conducted to assess the contamination of heavy metals in the water, sediment, aquatic plants and animals around a municipal landfill. The heavy metals were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES). The concentrations of cadmium (Cd), chromium (Cr), and lead (Pb) in water and sediment were Not detected (ND), 0.05 ± 0.98, and 0.02 ± 0.01 mg/L; and 0.47 ± 0.23, 18.65 ± 11.39, and 5.36 ± 2.08 mg/kg, respectively. A total of 24 aquatic plants from 4 species were collected from the municipal landfill. Cd concentrations in all plant species were within the standard, while Cr and Pb exceeded the standard. Forty-four fish from 4 species, 29 freshwater snails from 3 species, 10 freshwater prawns from 1 species, and 9 freshwater crabs from 1 species were collected. Cd and Cr concentrations in 2 species of fish samples exceeded the standard and Pb concentration in all fish species exceeded the standard. Cd and Pb concentrations in all freshwater snails were within the standard, except Cr. Meanwhile, Cd and Pb in freshwater prawns and snails were within the standard, except Cr. Five species of frogs were collected. Cd and Cr concentrations in 2 species exceeded the standard. Pb concentrations in all frog species exceeded the standard. The pattern of metal accumulation was fish > frogs > freshwater snails > freshwater prawns = freshwater crab (Cd), freshwater prawn > frogs > freshwater crab = freshwater snail > fish (Cr), and freshwater prawn > fish > frogs > freshwater snail > freshwater crab (Pb).     

Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein

As most cultivars of flax and linseed (Linum usitatissimum L.) are capable of accumulating cadmium (Cd), they are suitable candidates for phytoextraction of the metal from contaminated soils. In an attempt to enhance the phytoextraction capacity of L. usitatissimum through overproduction of an efficient heterologous Cd-binding peptide, we engineered linseed breeding line AGT 917 to constitutively express genetic fusion of α-domain of mammalian metallothionein 1a (αMT1a) and β-glucuronidase gus gene under the control of CaMV 35S promoter. An improved transformation protocol was developed, which involved co-cultivation of AGT 917 hypocotyl segments with partially removed epidermis with Agrobacterium suspension for 10 min in the presence of 200 mg l^?1 cellulase. The enzyme treatment increased the transformation efficiency (TE) 1.6-fold as compared to agroinfection without cellulase. Less pronounced impact on TE exerted 100 mg l^?1 acetosyringone, increasing TE 1.3-fold. When tested in soils amended with Cd at 20 and 360 mg kg^?1, the mature αMT1a::gus plants accumulated more Cd than parental AGT 917: the stem Cd concentrations in the best performing αMT1/2 line were 3.3- and 1.9-fold higher, respectively. Moreover, hypocotyl explants of αMT1/2 line showed 1.7-fold higher biomass than those of AGT 917 on media containing 15 mg Cd l^?1, indicating that αMT1a::gus did confer higher Cd tolerance to engineered plant. Overproduction of metal-binding peptides thus appears to be a viable strategy for the production of L. usitatissimum with improved phytoremediation capacity.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Molecular fractionation of Zn, Cu, Cd and Pb in the midgut gland of Helix pomatia L

Specimens of Helix pomatia were fed lettuce enriched with Zn, Cu, Cd and Pb for 30-35 days, after which time the distribution of the four metals in the midgut gland was studied by centrifugation, ion exchange and gel chromatography. In control snails the distribution between the soluble and particulate fraction of the midgut gland is relatively uniform, with a ratio of approximately 3:1 for Cu, Cd and Pb, and of 1:1 for Zn. However, after metal-loading about two-thirds of total Zn and Pb appears in the particulate fraction whereas little change in relative distribution is observed with Cu and Cd. After ion exchange chromatography of the supernatant both Cd and Zn eluted in one distinct peak, Cu in two peaks, whereas Pb seemed to be distributed unspecifically throughout the elution volume. If ion exchange is followed by gel chromatography, Zn, Cu and Cd are found associated with from one to four molecular species, ranging in mol. wt from 1000 to greater than 20,000, whereas no complex was found for Pb. The molecular patterns are specific for each of the three metals and there are some specific shifts in abundance, usually involving one molecular fraction, if control snails are compared with metal-loaded snails.