Toxicity of Heavy Metals and Biological Defense: Principles and

Since the beginning of the civilization ... Ca(II), whose size is similar to those of Cd(II), Hg(II), and .... organisms, fungi and bacteria, produce ...
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Toxicity of Heavy Metals and Biological Defense Principles and Applications in Bioinorganic Chemistry-VII Ei-lchiro Ochiai Department of Chemistry, Juniata College, Huntingdon, PA 16652 Some biologically essential elements have been discussed in the previous articles of this series (1-6).The present article discusses some adverse effects of metallic elements, particularly those of the so-called heavy metals. Heavy metals a r e ill-defined hut here a r e considered mainly cadmium, lead, and mercury. Copper, zinc, and silver also will be mentioned. Abundance and Toxicity A certain degree of correlation was shown to exist between the abundance in the environment and the level of elements present i n a biological tissue in a previous article (3).I t is to he noted (see Figs. 2 and 3 of (3))that the essential elements tend to be those that are relatively abundant in the environment. The organisms must have tried to utilize the elements that they inadvertently ingested from the environment. If the elements turned out to he useful to the organisms, they would have become essential to the organisms. Therefore, there is a better chance for a n element to become essential, if i t is present abundantly and in a readily accesihle form in the environment (the first basic rule of bioselection ( 1 ) ) . I t must he pointed out, though, that some elements such as Se and Co have become essential to organisms i n trace amounts despite their low abundances in the environment. No other more ahundant element could have substituted for these elements. On the contrary, a n ahundant element would not become essential, if i t is not useful to organims. "Aluminum" may be such a n element. Aluminum is uhiquitous i n the environment, though not necessarily i n a readily accesible form. How about the elements that organisms would encounter only rarely? They are the elements that are present a t low levels on the earth or in inaccessible forms. The heavy metals such a s lead, mercury, and cadmium belong to this group. Because most organisms have not had experience to deal with these elements, the organisms could he subjected to their adverse effects, ifthey ingested them. Some organisms, however, might have had enough experience to deal with the adverse effects of these elements over the long history of their evolutions. If that i s the case, those organisms must have developed some defense mechanisms against the adverse effects of those elements. Otherwise they would not have survived. In this sense, the elements that are rare on the earth tend to he toxic to the organisms in general. This is only a rough generalization. There are many exceptions, a s mentioned above. That is, some elements that are rare can become essential to the organisms, if they are critical to the functioning of the organisms. Natural versus Anthropogenic Discharge of Elements into the Environment Nature discharges a large quantity of a variety of the elements into the environment. The biosphere and its inhabitants collectively and/or individually have managed to cope with the natural discharge of elements. Otherwise the organisms would not have survived. Since mankind ap-

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Background Level AtOGght

Figure 1. The excess burden of several elements on the environmentlbiosphere; the blackened elements are considered to pose the greatest dangers; the shaded ones are likely to pose dangers. See the text for the meaning of the excess burden value.

2,Ionic radii: +Il=divalentcations in hioh-soin Fiaure " - . state (black circ e, or in on-sp n slate (&'I Ie c IC el, 4 1 ='wa en! cat ons n n gnSP n Slate oacn sqmre, or n loa-so n stalc ,bnllc sq-are,

peared, human activities have interfered with the natural activities. We have collected, hunted, cultivated, and had the pastures grazed by animals, and, a s a result, ravaged our environments. Since the beginning of the civilization and particularly since the industrial revolution we have been extracting the natural rcioul-ccr, utilizing them, and discharpln~ - - at lcast simx uortion of the utililed matcrlal into the environments, in such a way that they might interfere with the activities of the nature. Volume 72 Number 6 June 1995

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Figure 3. Radii of anions and oxyanions. Nature discharges elements through geological activities, weathering, and vulcanism. Their magnitude has been estimated. I t is more difficult to estimate the anthropogenic discharge level. One such estimate has been made by Nriagu (7). The ratio of "the total discharge (natural and antbropogenic) over the natural discharge" can then be computed. The ratio of "1.0" means no extraneous anthropogenic discharge; i.e., it represents the background level. The ratio higher than "1.0" means some extraneous discharge due to the mankind's activities and can be considered to be the "extra burden" on the nature and the biosphere. The ratio or extra burden values are shown in Figure 1. The figure suggests that we would have serious environmental problems not only with cadmium, lead, and mercury, but also with tin, selenium, molybdenum, and, perhaps, copper, arsenic, chromium, and nickel. Iron and zinc, though high in the ratio figure, may be tolerated better by the organisms, because they are essential and used by organisms in substantial quantities and, as a result, the organisms have better control mechanisms of the levels for these elements in their bodies. The situation mentioned above is that of'the average on thc global scale. It is ~ m s i b l ethat the local concmtration of acertain element ;auld be much higher than Figure 1 indicates. If so, the environmental problem there could be much worse than the figure suggests. Chemical and Physical Characteristics of Heavy Metal Cations The ionic radii of some metal cations of interest are shown i n Figure 2. The heavy metals are significantly larger than the more common cations. A n exception is Ca(II), whose size is similar to those of Cd(II), Hg(II), and Pb(I1). The variation in ionic radius among common transition metal cations is relatively small; i.e., within 510%. Some metallic elements also can exist often in the oxyanion forms. Figure 3 shows the ionic radii of more common oxyanions. The variation in size among them is even smaller than that among cations. The variation among similar oxyanions is less than 5%. Another important character relevant to the the biological behaviors would be the binding to ligands. Ametal cation prefers a type of ligand over another. Figure 4 shows the stability constants or solubility products for several interestingligands. The solubility product may not necessarily reflect the binding strength, but it has a lot to do with it. Pb(I1) forms a very insoluble phosphate salt. The major contributor to this fact is the size of Pb(I1). Its large size matches that of PO4> better than the other cations (radius-ratio effect).

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Figure 4. Log Kvalues: a = ethylenediamin complex formation, b = oxalate complex formation, c = complex formation with NHzCHzCHzSH, d = complex formation with HSCHGHzNfor phosphate. (CHZCOOH)~, e = Kspfor sulfide, f = Itp I t is apparent that Hg(I1) has a n inordinately strong affinity toward SZ. The heavy metal cations Hg(I1) and Pb(I1) are considered to be "soft": whereas. most of the transition metal divalent catlons listed (Fig. 2-41 are considered to bc on the borderline between tht. "hard" and the "soft". A "soft" acid prefers to bind to a softer base such as S2-. Hg(I1) is one of the softest cations. The soft metal can also form a stable o-bonded organometallic compounds. Typical examples are Hg(CH3I2,Hg(CH3)+and Pb(C2H&., Pb(CzHd3'. Selectivity in the Uptake of Elements

The environmental problem due to the extra burden on the biosphere would not arise, if the organisms have efflcient means to cope with the intrusion of excess quantities of the elements. There are two levels in the coping mechanisms. The first level of protection is to ingest or take up the elements discriminately, so a s not to take up unnecessary elements andlor unnecessary quantities. The second level of protection or defense is to render the adverse effects of the elements minimal when the organisms have ingested them. Some mechanisms of the latter type will be discussed later. In this section we will look a t the selectivity in the uptake mechanisms of elements. Metallic elements can be taken up either a s a cation or a s a n oxyanion. Exceptions for this general rule include the absorption of a metal-chelate a s a whole, as in the case of Fe(II1)-siderophore(see below). Cell membranes are virtually impermeable to metallic cations and oxyanions. Uptake of a n element, or any substance for that matter, ultimately depends on its binding to a biomolecule (acceptor, carrier protein etc). Cations and oxyanions are rather featureless. The only distinguishing factors that organisms can take advantage of are (a)the electric charge, ib) the size, ic) the preferred ligands, and id) the preferable coordination structure.

I n contrast, organic compounds may be easier to distinguish, as they have distinct structures, including the chirality. Because the variation in size among oxyanions is relatively small, anions of the same geometrical structure and of the same electric charge are difficult to distinguish. I t can be inferred that the hysiological receptorlabsorption site for the essential SO4 may take up such ions a s Se04'and Moo4% (8)and also that the regular uptake mechanisms for Pod3- could absorb V04%and AsO~~-, too (9).If a n absorption mechanism depends on a chemical reaction, rather than a mere binding, a more effective discrimination may be accomplished. But no such mechanism is known except for B02-, which can be bound to a carbohydrate moiety; then the B02-sugar moiety is taken up. The variation among typical divalent cations is not very large either. Thus, the selectivity in uptake cannot be expected very high, if it is based on the size and the electric charge alone. I n fact, not many efficient systems have been developed in the organisms to discriminate many of the common divalent cations through the size difference. This does not necessarily mean that ions of different sizes do not behave differently. They do, but the difference may not be sufficiently large to be utilized for discrimination in uptake. I t needs to be pointed out, however, that organisms do have a developed group of compounds that discriminate cations such a s Na(I), K(I), Ca(II1, and Mg(I1) according to size and electric charge. These compounds called "ionophores" (10) have, in general, cyclic structures, which have holes that match closely with the sizes of cations. Some organisms, fungi and bacteria, produce and secrete Fe(II1)specific chelating agents called "siderophores" (11).The entire chelates, rather than Fe(II1) ion, are then taken up by the organisms. There are also ion-specific channels that discriminate ions by their sizes. For example, a Na(1)channel hardly allows K(1) ions to go through. These are exceptional and very selective cases. There are a t least two different iron-uptake mechanisms in the human intestine. One of them absorbs Fe(I1) actively. I t has been demonstrated that Co(I1) or Mn(I1) competes for this Fe(I1)-absorption site (12). The selectivity of this site is not very great, most likely because Co(I1) and Mn(I1) have sizes similar to that of Fe(II), and thus can bind the Fe(I1)-binding site, though with different affinities. That is, this uptake mechanism cannot discriminate Fe(I1) against Co(I1) and Mn(I1) very effectively. This situation appears to be quite general a s far a s cation ahsorptions are concerned. Cd(II), Hg(II), and Ph(I1) could very likely enter a cell through the mechanisms to absorb the essential cations such as Ca(II), because of their similarity in size and electric charge. These heavy metal cations, however, may not be able to enter efficiently through the Fe(I1) or other essential smaller cation-absorption sites, for their sizes are sufficiently different. Other factors, i.e., "preferred coordination structure" and "preferred coordinating ligand atom", may be utilized to enhance selectivity in the cation binding. For example, the earlier transition metal ions prefer "O-atom" over "Natom" as the ligand, while the later transition metal ions tend to bind "N-atom'' ligands better, and the heavy metal cations bind with sulfur strongly. Unfortunately, these factors are rather subtle and have not been utilized fully by organims to enhance the selectivity. It might be pointed out, though, that the strong affinity toward sulfhydryl groups is employed by a special protein, metallothionein, to store or control the level of heavy metals including cadmium, mercury, copper, and zinc (see below).

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Another important factor is a kinetic one. Asubstitutioninert ion such a s Cr(III1, once bound to a biomolecule, cannot be displaced readily by another ion. This not only would lead to a high selectivity, but also it hinders the subseauent reaction. which mav reauire release of the ion from the biomolecule. ~ubstftutickinertcations, Cr(III), Co(II1)(in low-spin), Ru(I1) and Rh(II1) are not used widely in biological systems. Exceptions, however, include lowspin Fe(I1) complexes, vitamin Bl2 coenzyme (Co(III)),and, perhaps, Cr(II1) used to maintain a specific structure of DNA. The selectivity in cation-uptake, thus, is not very great in general, a s far as a single step-uptake is concerned. The cation uptake in single-celled organisms, therefore, may not be very discriminatory. The selectivity can be improved in multicellular oreanisms. because a metal ion must eo through several barriers before i t reaches a target c&. Suppose that two metal ions MI and M2 compete for the same binding site a t each step, and that the discrimination ~ that there are factor (K,/KJ is 10-to-one a t each s t e and three ba&iek to go through. Then the overall discrimination factor would have become 1000-to-one a t the target cells. A step or two in the process, however, often utilizes factors other t h a n thermodynamic ones. For example, a chemical reaction may be used, which enhances the selectivity enormously. For example, Fe(I1) and Co(I1) can be absorbed by an active transport mechanism in the intestine. When they come out to the portal vein side, they need to be oxidized in order to be bound to the transport protein, transferrin. This process is catalyzed by a copper-enzyme, ferroxidase; Co(I1) cannot be oxidized to Co(II1) and hence, cannot be bound to transferrin. Thus, a t this stage, the cobalt would be left behind and would not be carried to the bone marrow or the liver. Therefore, the overall iron selectivity over cobalt would be enhanced greatly. There is a verv. s~ecific mechanism for the transoort of cobalt-con. ta~ningv~raminB12(cobalamins~ Thr di.xussions nbove a ~ ~tol the v cases of metallic ions (aquo species). Some elements, particularly the heavy metals. can readilv form stable orpanometallic com~ounds. FO; example, mercury can form dimethyl mer&ry or monomethyl mercury, (CH3I2Hg o r CH3Hg(OOCCH3). These compounds can go through cell membranes more readily than divalent aquo-cations, because of their affinity to the membrane. Such a compound can be taken up almost indiscrimimately. Toxlcities of Heavy Metals The symptoms of the toxic effects of heavy metals may vary widely a t the physiological level, but the basic toxicity mechanisms a t the molecular level may be limited. The toxicities of heavy metals may be caused by the following mechanisms: 1. Blocking the essential functional groups of biamalecules

such as enzymes: Specific amino acid residues, such as serine-OH, eysteine-SH and histidine-N often constitute the active sites of enzymes. Hg(II), for example, binds strongly cysteine-SH's,blocking an enzymatic activity. 2. Displacing essential metal ions from biomolecules: Ameta1 ion mav disolace a native ion. if its afinitv to the bind-

eially enzymes and perhaps polynucleatides: A coordination of a cation may change the conformationof a protein, rendering it nan-functional. 4. Disrupting the integrity of biomembranes: Ametal cation may bind the negatively-charged head(s1of phospholipids and the integral protein residues of the membrane. 5. Modifying some other biologically active agents: For example Cd(I1)and PMII) appear to potentiate the endotaxVolume 72 Number 6 June 1995

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ins produced by bacteria (13).This might be due to their effect to .block .-. some enzymes which degrade the endotoxm, as m ( 1 ) . 6. Binding with bioanions, resulting in a decreased level of essential bioanions, especially P043-or a displacement of from biaminerals:F~~example,P~(II), an essential having a size similar to that of Ca(II),could replace Ca(I1) in a bone mineral. AS a result the mechanical strength of the . .hone mav " be affected.The size and the electric charee \rould he a n irnpurtnor fnrtor in rhese rfrects.One ufthe basic toxic rffect.i ut I'b 11, is considered 01t w bmding uf PO,,&, rendering its cytoplasmic level very low. ~~~

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These toxicity mechanisms are all based on the strong binding abilities of these metallic ions. And a s in the case of selectivity in uptake mechanism, substitution of a metallic ion by another is relatively simple. This is so except for substitution-inert metals. In practice, however, any metal cation bound to a large, somewhat rigid hiomolecule is kinetically difficult to displace, though not necessarily substitution-inert. This does not necessarily apply to the cases of proteins of a rather flexible conformation, as in the case of metallothionein discussed later on. What happens an apo.pro. often is that a replacement takes place k i n hinds a cation. A wrong cation can he incorporated in this stem if vresent there. Cd(I1) and Hg(II), for example, can replace the native Zn(I1) ion from many proteins and enzymes to a degree that depends on their affinities. Cu(I), M I ) and Au(I) also have similar characters to Zn(II1, Cd(II), and Hg(I1) because of their iso-electronicity. I t would he needless to point out that other cations such a s Cu(II), Ni(II), etc. also may displace a native cation such a s Zn(I1) from enzymes and proteins. For example, the carcinogenicity of Ni(I1) is considered to he due to its replacement of Zn(I1) and/or Me(I1). Because ". ., the essential cations in DNA oolvmerase. . " of its different size, Ni(I1) seems to increase the chance of binding wrong nucleotides, thus resulting in the formation of DNA of a wrong sequence (15). Many symptons of Cd(II) toxicity are believed to arise from the displacement of Zn(II) by Cd(I1). An example is the testicular necrosis by Cd(I1). Testes produce sperms. Hence, the DNA polymerase activity is quite high. DNA polymerase is a Zn(I1) enzyme, whose activity can be jeopardized by substitution by Cd(I1). (See ref. (14) for a list of enzymes affected by Cd(I1)). H d I I ) shows a n enormous affinity toward sulfide a s seenrn Figure 4. Because the cystei&c sulfhydryl group play a rntulytic role in a number of mzymvs, their eruymatic actlvltles are susceptihlr to IlgrII,. Or the binding of Hg\Il, to amino acid residurs can change a protein contbrmation sufficiently to render it inactive. Kxamples of enzvmes suscc:ptible to Hg 11, include NR/K-A'~I'as(: (the 50called s o d i i m pump); alkaline phosphatase, lactate dehydrogenase, and glucose-6-phosphatase (14). Ph(I1) also inhibits a variety of enzymes. However, the most interesting is its inactivating effects on the enzymes involved in the heme synthesis (16). Particularly important are ALA dehydratase (ALA-D) and ferrochelatase (FC). [ALA= baminolewlinic acid]. ALA-D, also called "porphohilinogen synthase", is a pivotal enzyme in the heme synthesis, and FC catalyzes the insertion of Fe(I1) into protoporpyrin. The inhibition of ALA-D results in a n increased level of ALA in urine, which is considered to be a sensitive measure of lead poisoning. The level of the nonfuctioning metal-free or Zn(I1)-porphyrin will increase when FC is inhibited. I t also has been reported that the glohin protein synthesis is affected by lead (17). The toxicities of h e a w metals have been reviewed extensively in references 14 and 18.

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Biological Defenses against Heavy Metals

When confronted with the toxic effects of heavv metals. the organisms had had to develop some means to defend themselves against them, As mentioned earlier, not all organisms have encountered all the heavy metals equally. The that were the metals did have developed mechanisms to combat them. 0 t h organisms mav not have had enoueh to deal with the .. ex~erience . heavy metal.;, and thus have only relatively inefTecti\x? mtt:tns to cornhat them. The extraneous anthro~ogen~c dlscharge of the heavy metals into the environment a s outlined above thus poses a serious problem. We will briefly explore some of the biological defense mechanims against heavy metals. m i s is not meant to be an exhaustive survey, but i t is given to illustrate some chemical p,.inciples behind the biological defense mechanisms, General Tolerance

Most elements seem to he tolerated by organisms to certain limited degrees. The degree of tolerance is highly dependent on the organisms, their life stages, a s well as on the elements. This is rather non-specific. Metal ions are to be confined to locales that have little influence on the genera1 biological activities. Such places include cell wall, some noncritical portions of the cell membranes, vacuoles, lysosomes (191, hard tissues such as bone, and extra body tissues such a s hark and hair. As long as the metallic ions are confined in these non-critical places, they may not exhibit their toxic effects. Obviously the extent of tolerance by these means is limited and highly dependent on the structures of the organsims. The toxic symptoms will manifest when a metal ion level exceeds a certain threshold level, unless other more specific defense mechanisms are available.

secretion o f ~ u c u sMaterial This is found especially in fish. Mucus consists of anionic polysaccharides, such a s chondroitin sulfate, ascophyllan, and fucoidan. These contain carhoxylate and/or sulfate groups, and they bind cations, It has been that fish killed by a high level of Pb(I1) or Zn(I1) were found to have high levels of mucus around their gills (20). I t has suggested that mucus may function as a defense mechanism against cations. Metallothioneins

Metallothioneins (MT) are low molecular weight (670001, cysteine-rich proteins t h a t bind metals such a s Zn(II), Cd(II), and Hg(I1) (23). MT and its analogues are widely distributed among organisms, from bacteria and fungi to plants and mammals. A typical mammalian (Zn, Cd) MT is folded into two domains that form separate polynuclear metal cysteine thiolate clusters; Cd4-Cysll cluster in the C-terminal domain and Zn3-Cyss cluster in the N-terminal domain. This structure has been determined hv X-rav crvstalloera~hv(21). and a m e a r s to be commonto most MTS whiih h i d seven metaiiic ions. Metallic ions that form M7-MT include Co(I1). Ni(I1). HdII). Ph(II), and Bi(II1) in addition to Zn(I1) and Cd(11)'i22): Ag(1) and CuU) can displace Zn(I1)or Cd(I1)from a MT and bind in the form of MIS-MT(22). Hg(I1) then displaces 12 Ag(1) or Cu(1) ions and form Hg7-MT (22). It has been demonstrated that synthesis of MT can be induced by CdUI), Zn(II), Hg(II), and &(I) (23). This fact indicates, a s suggested hy many researchers, that MT could function a s a protective means against toxic elements such as Cd(II), Hg(II), and M I ) . Zn(I1) is an essential element (3). and MT also is involved in the control of Zn-metabolism. The chemical basis of these functions is

obviously the strong affinity of these ions toward thiolate. 'Ibxic metals, thus, bound to MT cannot exhibit their adverse effects. However, lowering pH of the medium or other yet-to-be-identified mechanisms can release the metal ions from MT, and hence, MT may control their cytosolic levels, and also may function a s a temporary storage for certain elements such a s Zn(I1). The metabolism of MT is not very well understood. However, it has been reported that Cd-MT is degraded rapidly in kidney cells, releasing Cd(II), which is considered to be the cause of the proximal tubular damage (24). The situDresation in liver seems to be different. For exam~le. . . the . ence of MT decreases significantly the release of metals (Cu and Zn) into bile.. suggesting -- that the metals remain chelated to MT.

shown to inhibit Cd-induced destruction of nonowlating ovaries and placenta, and has protected against the teratogenic effects of cadmium (30). I t is interesting to note that the retention of Cd by tissues actually is increased in the presence of selenium. A similar effect has been observed in reeard to selenium versus mercurv (30). Tuna and bonito tend to accumu1:itr mercury to a high level: vet the fish thtmst!lve.i art: h t ~ l t h vThe tuna =,as found to contain also a high level of selenLm, and the HgISe mole ratio was close to one-to-one (31).Subseauentlv. a linear relations hi^ also was reported for Hg versus ~e'contentsin seals and other marine mammals (32) a s well a s mercury-mine workers (33).

Metallothioneinlike Compounds

A dense granular structure (lead inclusion body=LIB) was first discovered in the nuclei of renal tubular lining cells and hepatic cells of children dying of acute lead poisoning (34). LIB is observed a t smaller lead dosages, even before a n increased excretion of ALA ensues (see above). Similar inclusion bodies have since been reported in other animals (34) and also a moss (35). The LIB isolated from the kidneys of poisoned rats contained a n average of 57 m m (fresh basis) of lead. The whole kiduevs of the uoi&ned rats contained 0.8 ppm, while the levil of Pb inAthe control kidnevs was 0.009 m m . The LIB consists of a dense central core and an outer Kbrillar zone. The protein of the LIB has a high content of aspartic acid, glutamic acid, glycine, cysteine, and tryptophan. The lead in LIB can be removed readily in vitro by EDTA. I t has been suggested that the formation of LIB serves as a protective mechanism against lead poisoning (34).

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Lead Inclusion Body

The descriptions above apply to the mammalian type of MTs. Other organisms produce MT-like proteins that are not exactlv the same a s the mammalian MT. For examole. a yeast copperthionein can be induced by Cu(II), but noi b i Zn(I1)or HdII). I t is distinct from the mammalian MT. but the 12 cysteine residues are conserved. The protein protects cells against comer uoisoning. but is auuarentlv dis.. pensable foqnormal ielluiar grow& (25). Manv authors had r e ~ o r t e dthat Pb(I1) did not induce MT, but Ikebuchi et al.in 1986 reported that a MT-like protein containing Pb(I1) was induced along with a Zn(I1)MT in the rat liver when lead acetate was administered (26). It appeared to be very similar to the Zn-MT(II), but distinct in electrophoresis. It still is not clear whether the Pb-MT was induced by Pb(I1) rather than Zn(I1) which may have contaminated the lead acetate sample, though Conversion to Readily Excretable Forms its zinc content was reported to be less than 0.05%. The imuortance of a MT-like rote in in detoxifvin~ . ., Pb(I1) has A single-celled organism is small in size, and hence, if a not been well estnhlinhrd, though PO 11, does tnnd lo a prechemical entity can be changed to a volatile form, it can fmmvd MT a s nientloned nbow Pbt 11, mav not induce the readily diffuse out of the cell. Hg(I1) can be converted into synthesis of MT, not because it cannot bind to MT or a facHg(0) (36) or (CH&Hg (37)in some bacteria. Both of these tor to induce its synthesis, but rather because Pb(I1) may forms of mercury are much less toxic t h a n Hg(I1) or be trapped by phosphate entities present in vivo before it CH3Hgt, a s well as they have significant vapor pressures. reaches the MT synthetic a .~.o a r a t u s . Methylcobalamin has been demonstrated to effect the Cadmium-resistant Pseudomonos putido produces methvlation of Sn(I1. IV) and Pb(I1. N)a s well a s He(I1) three Cd!Il,-binding proteins: 3815,7216, and 7239 in molecular weight (271.-fhey contain six to eight cysteine residues and bind cadmium, zinc, and copper. Coordinating liis~reahily + excretable fense mecLanism, a s ? c H ~ ) ~ A gands seem to be cysteiue residues,-histidine nitrogen, through the kidneys (9). and, perhaps, glutamate carboxylates. Suspension cultures of Rauuolfia serpentina (a higher Concluding Remarks plant) produced heavy-metal complexing peptides when It has been shown that the toxic effects of and the biogrown in a medium containing CdS04 (28). The peptides logical defenses against heavy metals can be understood were identified to be (y-glutamate-cysteine),-glycine(n=2 basicallv in terms of the fundamental chemical urinci~les to 11). The composition suggests that the peptides are the and tbebasic properties of these elements. I t also has been derivatives of glutathione. The peptides, called "phytoarmed that the toxicities of rare elements are caused uarchelatins". were shown to be induced also bv Cu(I1). HdII). Pb(II), and Zn(I1). The induction of the peptides b i C ~ I I ~ tially by the fact that the organisms rarely have encountered and, thus, have not developed effective means to also was observed in cell cultures of other ~ l a n t including s combat with them. Bereris stolonifera, Fumaria paluiflora, and Galium mollugo. Literature Cited 1. Oehisi. %I. J. Chem. Educ 1978.55.631633. Sulfide and Selenide 2. Ochmi, E-I. J. Chem.Educ. 1987,64,942944. 3. Oehiai. E-I. J. Chpm Educ 1988.65.943-946, One copper-detoxifying mechanim found in many fungal 4. Ochlai, E-I. J. Chem Edlrc. 1991,68. 10-12. species is the excessive production of HzS. For example, 5. Oehiai. E.-I. J. Chem Educ 1991.68.627-630. 6. Ochiai. E-I. J. Chem Educ. 1993, 70. 128-133. certain strains of yeast acquire a brown color when cul7. Ntiagu, J. 0. Enuimnmsni 1990.32 171, 7-33. tured in the presence of copper, due to the formation of in8. Schneider, E. G.; Durham. J. C . . Sacktor. B.J. Eiol. C h z m 1984.259.14591-14599. 9. Fowler, B. A. In Toxicology o/ Doc< Elements; Coyer, R. A,: Mehlman, M. A,. soluble and hence inactive CuSICuzS (29). Microscopic Eds.: Hemisphere: N e w York.1977, pp 79-122. studies have shown that the copper sulfides were located 10. Simon, W.; M o l t W. E.; Mcicr,P C.StrucI. Bondg. 1973,16,113:Oehiai, E.-I. Bioimainly in and around the cell wall. nor~anicChrmistw, An Intmduetion; Allyn and Bseon: Boston. 1977. pp422428. 11. Neiiands. J. B. Sirucl. Bondg. 1972, 11, 145; Hidel R. C. Slrub. Bondg. 1984. 58, An antagonism between selenium and heavy metals has 25-87. long been known. For example, selenite has a protective 12. Thomson, A. B. R.; Valberg, L. S.; Sinclair D. G. J. Clin Invert. 1911. 50, 23842394: Thomson, A. B. R.: Olatunbosun. D. A,:Valberg. L. S. J Lob. Clin. Inmest. property against Cd-toxicity in testis necrosis, has been

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