Toxic Heavy Metal

Heavy Metals

Most of the references on potential mechanisms of plants-mycorrhizae-xenobiotics interaction are again related to heavy metals. It is generally acknowledged that mycorrhizal amelioration of metal toxicity is achieved via several physiological processes rather than through a single mechanism and that participation of single mechanisms in metal detoxification is metal- and species-specific 4, 95, 96 . As the main mechanism, immobilisation of HMs in both intra- and extraradical fungal structures and resulting restriction of metal transfer into plant tissues has been suggested 4, 98 . In accordance with this theory, it was shown that inoculation with different EcM strains decreased zinc and cadmium uptake into pine shoots and the fungus producing more extensive extraradical mycelium showed greater effect on overcoming metal toxicity and lowering shoot metal concentrations 50, 54 . Non-mycorrhizal pine seedlings were reported to transport more copper to above-ground parts, however, roots...

Initial considerations

Buffer composition is frequently critical for successful enzyme purification. Factors such as pH, ionic strength and nature of the buffer can all contribute to protein instability. Although selection of the optimal buffer composition is often an empirical process, some rational judgements can be made. For example, cysteine proteinases generally require the inclusion of a sulphydiyl reducing agent such as DTT or 2-mercaptoethanol in the buffers. Because heavy metals will readily inactivate cysteine proteinases, it is essential that the highest quality water and reagents be used for buffer preparation. As an added precaution, it is a good idea to also include EDTA. It should be noted that some serine proteinases and even metalloproteinases contain a sulphydiyl group whose integrity is necessary for expression of enzyme activity. Therefore, inclusion of thiol-reducing agents in buffers is not always limited to cysteine proteinases. For example, prolyl oligopeptidase (EC 3.4.21.26), a...

General scheme for purification of proteolytic enzymes

The initial stages of proteinase purification generally contain one or more crude fractionation steps to remove bulk protein and prepare the material for subsequent chromatography. Pilot studies must be performed to determine which procedure is most suitable and the experimental conditions to be used. The most commonly employed initial step is ammonium sulfate fractionation (34). Ammonium sulfate of high purity designed for protein purification should be used, since trace contaminants of heavy metals can inactivate many proteinases. Batch ion-exchange chromatography is a reasonable alternative provided that the protein of interest binds to the exchanger. In this method, the ion-exchanger is equilibrated with the same buffer used to prepare the tissue extract, mixed with the extract and the suspension poured into a B chner funnel. Unbound protein is removed by washing with the equilibrating buffer until the absorbance at 280 nm of the effluent reaches baseline. The protein of interest...

Biosensors Sensing the Environment

Firefly luciferase-engineered bacteria, cyanobacteria, yeast, algae, plants, and multicellular animals have been successfully used as whole-cell biosensors 78-84 . Because firefly luciferase depends on ATP, its bioluminescent signal can be conveniently linked to the energetic status of the cell. In the simplest cases these cells can be directly used in general toxicity tests. More sophisticated bioassays for specific toxic agents have been constructed by fusion of a luciferase gene to a stress-inducible transducer, the luminescence being directly related to the degree of activation, detecting down to ppm amounts of these compounds. The latter are exemplified by whole-cell biosensors for heavy metals such as mercury and arsenic, phenols, agrochemicals, hormones 84 , and other environmental disruptors. They can also be used as reporter genes to monitor the bioavailability of nutrients such a nitrogen and iron.

Heavymetal derivatives

The historical introduction (Chapter 1) has already described the early use of high concentrations of salt employed empirically in the salting process as a preservative for meat, and the use of copper and silver vessels to prevent water from becoming fouled by microbial growth. Salting is still used in some parts of the world as a meat preservative and salts of heavy metals, especially silver, mercury, copper and, more recently, organotin, are still used as antimicrobial agents. The metal derivatives of copper, mercury, silver and tin, which find use as antiseptics and preservatives, will be discussed in this chapter. Kushner (1971) has reviewed the action of solutes other than heavy metal derivatives on microorganisms.

Eukaryotic Luciferases 8221 Firefly Luciferase

The bioluminescence resulting from firefly luciferase exhibits the highest quantum yield known for a bioluminescent reaction (0.88, which is about 10 times larger than that of bacterial luciferase) 1 . Like bacterial luciferase, mammalian organisms do not exhibit endogenous firefly luciferase activity. However, the genes required to synthesize the substrate (luciferin) are not present thus, addition of an exogenous substrate is always required for bioluminescence to occur. There has been controversy in this matter because the substrate cannot be permeated into the cell 25 . The addition of solubilizers such as DMSO has been required to facilitate entry into the cell. An interesting feature of some eukaryotic luciferases is that they can exhibit light in different wavelengths, ranging from 547 nm to 593 nm 26 . This characteristic has been employed only in colony detection on the same plate, but it can be further exploited in multi-analyte detection by fusing...

Environmental Pollutants

A variety of bacterial whole-cell sensing systems based on the promoter-reporter gene concept have been developed for the specific or selective detection of single pollutants or classes of pollutants. Many microorganisms have evolved the ability to survive in suboptimal conditions, including contaminated environments. Such ability usually relies on genetically encoded resistance systems. In the presence of toxic compounds such as heavy metals and metalloids, particular bacteria can synthesize specific proteins that confer resistance to those substances. The mechanisms of resistance vary. Some microorganisms develop efflux pumps, loss-of-uptake systems, or chemical detoxification systems 61, 62 . Other bacterial strains living in contaminated sites can degrade organic xenobiotics and utilize them as carbon and energy sources 63 . Many of these resistance pathways are inducible, meaning that protein synthesis occurs only as required by the presence of given compounds, which makes their...

Arbuscular Mycorrhizal Fungi

There are relatively scarce data on the effects of soil contamination on field abundance and diversity of populations of AM fungi. It was found that contamination of soils with heavy metals decreased numbers of AM propagules and reduced mycorrhizal infectivity of soils as compared to uncontaminated sites 15, 16, 17 . Nevertheless, spores of the AM fungi and relatively high levels of root colonisation were reported even from highly contaminated mine spoils as well as polluted agricultural soils 18, 19, 20, 21, 22 . A great variability in HM tolerance of different AM fungi isolates have been shown 23, 24, 25 , with higher HM tolerance often reported for indigenous isolates obtained from polluted soils as compared with non-indigenous ones 26, 27, 28 . Especially the isolates capable to tolerate extreme soil contamination represent a valuable material for potential inoculation of plants within phytoremediation programmes. More attention should be, however, paid to the stability of high HM...

Ectomycorrhizal Fungi

Negative effects of heavy metals on the development of EcM symbiosis have been repeatedly reported, both in vitro as well as in association with host plants. Decreasing abundance and diversity of Ectomycorrhiza morphotypes with increasing concentration of heavy metals was observed in a vicinity of the fertiliser factory 42 . Similarly, lower average number of EcM root tips in Pinus sylvestris was reported from site polluted with acid rain and with high aluminium availability than from a reference unpolluted site 43 . On the contrary, no evidence for a reduction of the genetic variation of subpopulations of the EcM fungus Suillus luteus caused by HM contamination was revealed in another field study 44 . High chromium and nickel concentrations were reported to decrease EcM colonisation by Pisolithus tinctorius in Eucalyptus urophylla 45 . Five different heavy metals were shown to negatively influence nitrogen acquisition efficacy of ectomycorrhizal birch seedlings 46 . On the contrary,...

Ericoid Mycorrhizal Plants

Results of the studies confirmed mycorrhizal colonisation with the ErM fungi as an important factor positively influencing the resistance to heavy metals. Ericaceous plant species themselves display HM resistance 81 , but it is supposed that successful colonisation of certain HM polluted sites requires adaptations from both host plant and ericoid mycorrhizal fungus 12 . In general all reports on experiments with the ErM fungi showed lower content of heavy metals in shoots of mycorrhizal plants as compared to non-mycorrhizal plants but often increased HM accumulation in the roots. Seedlings of Vaccinium macrocarpon colonised by Hymenoscyphus ericae showed increased tolerance to lead expressed in reduced translocation of Pb to the shoots when compared to non-mycorrhizal seedlings 38 . In another study, two races of Calluna, one from a HM polluted site, the other from an unpolluted natural heathland, were compared under mycorrhizal and non-mycorrhizal conditions in sand cultures with...

Ectomycorrhizal Plants

On the other hand, some studies report that EcM fungi do not limit uptake of heavy metals by their host plants, moreover, they can increase such an uptake, which was the case of zinc in Pinus sylvestris inoculated with Thelephora terrestris 54 . Increased uptake of aluminium by host plants mediated by EcM fungi was show as well 90 . EcM fungi can affect HM stress in host plants differently according to the metal screened Laccaria proxima, Lactarius hibbardae, L. rufus and Scleroderma flavidum increased tolerance of Betula papyrifera to different concentrations of nickel but negative growth effect of ectomycorrhizal fungi were observed at elevated levels of copper 91 . Also the concentration of the metal in the medium plays a significant role regulation of zinc uptake by EcM fungi in Pinus sylvestris was dependent on the concentration of metal in the mycorrhizosphere 92 . At low Zn concentrations, the EcM fungi increased its uptake, whereas at high external Zn concentrations, the EcM...

Assessing Risks And Containing Or Mitigating Gene Flow Of Transgenic And Nontransgenic Phytoremediating Plants

Plants have been used to correct human error over the ages. The few species capable of revegetating Roman lead and zinc mine tailings in Wales 2 taught us that there are a limited number of species that can withstand toxicants some by exclusion, and others that can withstand toxic wastes after they have been taken up. Plants with the latter type mechanism are of interest for phytoremediation. Ideally, one might consider that it is best to use the species that naturally take up particular toxic wastes, but these are often slow growing (e.g. mosses, lichens, or the Thlaspi species that take up heavy metals) 3 or may have a potential to be weedy. If the desired wild species do not exist locally, there may be a reticence or legal issues about introducing them into the ecosystem, toxic as it may be, due to fear that the plants or their genes may spread to other areas. Two types of multi-cut species are used, with the cut material burnt to extract the heavy metals or to oxidize the organic...

Yaolin Wang Bert W OMalley and Sophia Y Tsai 1 Introduction

The Tet repressor operator system (tTA) with tetracycline as an inducer (6,7,8). More recently, the direct fusion of a protein of interest to the hormone-binding-domain (HBD) of the steroid receptors has been shown to render the fusion protein responsive to steroid (9,10). For example, the GAL4-HBD fusion protein is capable of transactivating a target gene by binding to the GAL4 binding sites (17-mer) upstream of the target gene in the presence of hormone (11). Whereas these systems have been used in tissue culture systems to regulate gene expression, their use in gene therapy is limited for the following reasons. The use of heat shock promoter for gene therapy is obviously impractical, whereas heavy metals (such as Cd-Zn) and IPTG are known to be cytotoxic (4,12) and the induction is slow. Regulation of the protein function by fusion directly to steroid receptor HBD might change the conformation of the protein to be regulated. For this reason, the function of the protein after fusion...

Tolerance Mechanisms

The toxic effects of plant exposure to elevated metal concentrations, such as growth inhibition and chlorosis, are well documented. However, as for other organisms, the biochemical understanding of metal toxicity is limited. Class B metals such as Cu(I) and Hg and borderline metals such as Cu(II), Zn, Ni, and Cd (12) can form complexes with nitrogen and sulfur atoms in proteins, thereby potentially inactivating them. The nonessential metals Cd and Pb compete for binding with the essential metals Ca and Zn. Also implicated in symptoms of metal toxicity is the redox activity of some metals (e.g., Cu, Fe) that may result in the formation of reactive oxygen species. A complex network enables plants to control tightly the intracellular concentrations and distribution of essential heavy metals such as copper and to minimize the cytosolic concentrations of nonessential heavy metals such as cadmium. The interplay of mainly transport and chelation processes that constitutes this network...

Mechanisms Of Hypertolerance

Species and genotypes, which are part of the heavy metal plant communities established on sites enriched in toxic heavy metals, are necessarily hypertolerant. The molecular mechanisms underlying this trait are little understood. The typical plant response to metal exposure, the formation of phy-tochelatins (see earlier), appears not to be involved in naturally occurring hypertolerance. Silene vulgaris ecotypes differing in Cu, Zn, or Cd tolerance have been investigated thoroughly in this respect. When Cd-tolerant and -sensitive lines were compared, it was found that the sensitive lines form more PCs in their roots upon Cd exposure. Furthermore, synthesis of PC-Cd complexes is more rapid in sensitive lines and the PC composition is the same in sensitive and tolerant lines (120). Equivalent results were obtained for S. vulgaris ecotypes with differential Cu and Zn tolerance

Ericoid Mycorrhizal Fungi

For the ErM fungi, there are only few reports on how heavy metals influence development of symbiosis and growth of the host plants. Screening the effects of lead on the growth of ericoid mycorrhizal mycobiont Hymenoscyphus ericae cultivated in vitro showed that this fungus was able to grow on media containing up to 400 .ml-1 Pb 38 . The same fungus was able to grow over all iron concentrations tested (0-144 .ml-1), exhibiting greater resistance than its host plants 39 . The growth of another ErM fungus Oidiodendron maius isolated from mycorrhizal roots of Vaccinium myrtillus growing in heavily contaminated soil was investigated in the presence of zinc ions 40 and the authors found strong specificity of HM tolerance for each fungal strain. In the presence of increasing concentrations of Zn salts (especially at higher ion concentrations), better performance of the mentioned isolate was observed in comparison with isolates from unpolluted soils. Differential resistance was also observed...

Stephanie L Lara Stephen P Evanko and Thomas N Wight

Enhancement is achieved by staining with the heavy metals uranyl acetate and phos-photungstate and by rotary shadowing with evaporated metal. We have used this technique to evaluate the structure of individual proteoglycan monomers as well as proteoglycan aggregates from human intestine (5), cultured arterial smooth muscle cells (6), bovine aorta (7), and nervous tissue (8-10).

Environmental and Safety Issues on PVC

These metal compounds leaching out of PVC into the environment in landfill situations is a serious potential concern. However, in landfills (as well as in the potable water systems that use PVC pipes), these compounds are for the most part locked in the rigid plastic matrix. Unlike in plasticized systems, no significant rates of migration of these chemicals is unlikely. Consequently, release of heavy metals in any significant quantities into landfill leachate, sewer environments, or water distribution networks is not anticipated 23 .

Removal Of Pollutants Via Plants

68 Kamal, M Ghaly, AE Mahmoud, N and Cote, R (2004) Phytoaccumulation of heavy metals by aquatic plants. Environ Int 29 1029-1039 72 Fritioff, A and Greger, M (2003) Aquatic and terrestrial plant species with potential to remove heavy metals from stormwater. Int J Phytoremediat 5 211-224 80 Li, Q-F Li, Z-A Ren, H Du, W-B Tian, S-N and Peng S-L (2004) The role of wetland plants and soil in decontamination of heavy metals. J Trop Subtrop Bot 12 273-279

Schematic Diagram Of Basic Principles Involved In Lsab Immunoperoxidase Method

Tissue specimens must be fixed properly in order to attain adequate histologic sections. There are currently many types of fixative available for this purpose. The various types of fixative have different methods of stabilizing the tissues, thus resulting in different effects on the immunopathology process. Some of the commercially available fixatives are alcohol-based, while others contain heavy metals as the main operative component. However, virtually all diagnostic dermatopathology laboratories use a formaldehyde-based fixative for routine tissue fixation. These fixatives are readily available, relatively inexpensive, and provide good fixation for most tissues and clinical situations. As other fixatives such as mercury-based, picric acid, and alcohol-based fixatives are not widely used in der-matopathology, their inherent problems pertaining to immunopathology will not be discussed in this volume. For more specific information addressing these specific issues, the readers are...

Quarter Staggered Triple Helical Structure

Yet, later measurements indicated that the type I collagen molecule had numerous bends and was not completely rigid (23). Type II and III collagen molecules have slightly higher translational diffusion coefficients and slightly shorter end-to-end distances (23), suggesting that the later collagen molecules are more flexible than type I collagen. Results of recent modeling studies suggest that the types I-III collagen molecules are made up of alternating rigid and flexible domains that are conserved within the collagen fibril with types II and III collagen molecules being more flexible than type I (19). The flexible domains at first approximation coincide with the positively stained bands in the collagen D period observed in the electron microscope after staining with heavy metals (19,24).

Hairy Roots In Phytoremediation And Phytomining Studies

Cleansing Biotope Phytoremediation

Roots play a primary role in phytoremediation and phytomining, as they are the plant organs in direct contact with soil pollutants and heavy metals. Accordingly, there is a particular need to understand the biochemical and physiological functioning of roots in contaminated environments. Hairy root cultures are a convenient experimental system for such studies. In contrast to whole plants grown either in soil or hydroponically, hairy roots can be propagated indefinitely so that entire experimental programs can be carried out using tissues derived from the same plant, thus avoiding the effects of variability between individual specimens. Use of axenic conditions in hairy root culture prevents microbial symbiosis disguising the remediative activity of plant tissues, and better control over conditions at the roots can be exercised compared with soil cultivation. Separation of hairy roots from the leaves of plants also allows identification of the properties and functions of the roots...

Identification of Additives

Several techniques have been developed to rapidly identify additives in plastics as part of an overall plastics recycling operation. Some techniques such as Fourier transform infrared (FTIR) can combine resin identification with information of the presence or absence of additives such as flame retardants or talc fillers 49, 62, 65 . The sliding spark technique developed at the University of Duisburg in Germany can identify a range of heavy metals along with the type of resin 68, 69 .

Domen Lestan

Phytoextraction refers to the use of metal-accumulating plants, which are able to transport and concentrate inorganic contaminants, most importantly heavy metals but also metalloids and radionuclides, from the soil into their harvestable, above-ground parts. Metal-enriched plant biomass can be safely disposed of as a hazardous material or, if economically feasible, used for metal recovery. For phytoextraction to be possible, the metals must be in soil horizons within a plant's root zone, be bioavailable for plants and plants must have a genetic predisposition for compartmentalisation of extracted metals. Some metals are readily bioavailable for plants Cd, Ni, Zn, As, Se, Cu, and some have a low bioavailability Pb, Cr, U, Hg 1 .

Other Impurities

Chromatographic leachables such as Protein A, derived from the affinity resin used for monoclonal antibody purification, are generally measured by immuno-chemical methods. A number of ELISA-based methods are currently available for this determination, including kit-based methods. It should be noted that the best practice is to use an assay targeting the specific protein A type used in the antibody purification process, as a variety of antibody purification resins are currently available that use Protein A derived from natural or recombinant sources. For nonantibody products, chromatographic leachables such as heavy metals from IMAC columns can be measured using atomic absorption or ion-coupled plasma (ICP) spectroscopy methods.

Calcium

Large quantities of calcium can be deposited outside the protoplast, in cell vacuoles and in cell walls. Calcification strengthens plant cell walls and is thought to increase the resistance of a plant to infection. By forming insoluble salts with organic acids, calcium immobilises some potentially damaging by-products. The element gives protection against the effects of heavy metals and conveys some

Example 153

Activities and to take action to prevent spillage, leakage, or seepage of oil from the site. In April 1974, in response to evidence of recent oil dumping and overflowing, the Charleston County Health Department (CCHD) ordered the site closed. In March 1982, George Geiger purchased the site. In 1983, the lagoons were filled with local soils after approval was denied to excavate and dispose of the oil-contaminated soil. Subsequently, the site was used to store equipment. The primary contaminants of concern were arsenic, toluene, organics, PCBs, and heavy metals (lead, chromium, mercury).

Antioxidants

Selenium and heavy metals Metals such as arsenic, lead, mercury, cadmium and thallium are all quite poisonous. Furthermore, industrial uses of these metals have led to large-scale environmental contamination. One of these, cadmium, has been specifically implicated as a cause of prostate cancer. Selenium alters the toxicity of heavy metal ions in several ways.19'20 It is important to note that selenium can bind these metal ions into complexes that are insoluble in water. To a certain extent, selenium can bind to these metal ions in the gastrointestinal tract, diminishing the absorption of both selenium and the toxic metals. Once selenium has been absorbed, it can form complexes with these metal ions, lessening or delaying the toxicity of these metal ions and potentially reducing the risk of cadmium induced prostate cancer.

Pancreas

Metallothionein I is predominantly expressed in the pancreas and liver (and to some extent in the kidney) and it is powerfully induced by heavy metals, such as zinc. In the absence of any induction, these transgenic rats showed distinct signs of the activation of polyamine catabolism in the pancreas, such as greatly enhanced accumulation of putrescine and the appearance of A -acetylspermidine, yet the pools of higher polyamines were relatively well preserved in comparison with the wild-type animals (39). Nontoxic doses of zinc powerfully induced the promoter and resulted in a striking stimulation of pancreatic SSAT that led to an almost total depletion of pancreatic spermidine and spermine pools and massive accumulation of putrescine (39). No such changes were found in wild-type animals similarly exposed to zinc. In the transgenic animals, the activation of polyamine catabolism by zinc was accompanied by a rapid development (within 24 h) of acute necrotizing...

Sequestration

Hmtl from S. pombe was the first gene identified that encodes a protein involved in vacuolar sequestration of heavy metals (66). Hmtl was cloned on the basis of the complementation of a Cd-hypersensitive S. pombe strain lacking vacuolar sulfide-containing PC-metal complexes. The corresponding protein belongs to the large family of ABC-type transporters. It localizes to the vacuolar membrane and transports PC-Cd complexes and apo-PCs (67). S. pombe cells overexpressing hmtl accumulate more Cd2+ and are more Cd2+ tolerant. This indicates the potential of corresponding experiments in plants. However, no transgenic plants expressing hmtl have been described yet. Also, the respective ABC transporters that account for the transport of PC-metal complexes in plants are not yet known. The Arabidopsis MRPs 1-4 were shown to mediate the vacuolar sequestration of various xenobiot-ics conjugated to glutathione but lack PC transport activity (68). This led to the speculation that additional factors...

Thiol Status

The role of glutathione in cellular protection (figure 6.10 and see below) means that if depleted of glutathione the cell is more vulnerable to toxic compounds. However, glutathione is compartmentalized and this compartmentalization exerts an influence on the relationship between glutathione depletion or oxidation and injury. The loss of reduced glutathione from the cell leaves other thiol groups such as those in critical proteins, vulnerable to attack with subsequent oxidation, cross-linking, formation of mixed disulphides or covalent adducts. The sulphydryl groups of proteins seem to be the most susceptible nucleophilic targets for attack, as shown by studies with paracetamol (see Chapter 7), and are often crucial to the function of enzymes. Consequently, modification of thiol groups of enzyme proteins such as by mercury and other heavy metals, often leads to inhibition of the enzyme function. Such enzymes may have critical endogenous roles such as the regulation of ion...