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(201) Mikalsen, S.-0. (1990) Effects of heavy metal ions on intracellular communications in Syrian hamster embryo cells. Carcinogenesis 11, 1621-1626. (202) Alcedo, J. A,, and Wetterhahn, K. E. (1990) Chromium toxicity and carcinogenesis. Int. Reu. E x p . Pathol. 31, 85-108. (203) Doll, R. (1984) Nickel exposure: a human health hazard. In Nickel in the Human Enoironment (Sunderman, F. W., Jr., Ed.) IARC Scientific Publication 58, pp 3-11, Oxford University Press. (204) Robertson, F. M., Beavis, A. J., Oberyszyn, T. M., O’Connell, S. M., Dokidis, A., Laskin, D. L., Laskin, J. D., and Reiners, J. J., Jr. (1990) Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-0tetradecanoylphorbol 13-acetate. Cancer Res. 50, 6062-6067. (205) Frenkel, K., and Gleichauf, C. (1991) Hydrogen peroxide formation by cells treated with a tumor promoter. Free Radical Res. Commun. 12-13, 783-794. (206) Cross, C., Omaye, S., Rifas, D., Hasegawa, G. K., and Reddy, K. A. (1979) Biochemical effects of intratracheal instillation of cadmium chloride on rat lung. Biochem. Pharmocol. 28,318-388. (207) Katsnelson, B. A,, and Privalova, L. I. (1984) Recruitment of phagocytizing cells into the respiratory tract as a response to the cytotoxic action of deposited particles. Enuiron. Health Perspect. 55, 313-325. (208) Lynn, W. S. (1984) Control of the cellular influx in lung and its role in pulmonary toxicology. Enuiron. Health Perspect. 55,
307-31 1. (209) Nieboer, E., Tom, R. T., and Rossetto, F. E. (1989) Superoxide dismutase activity and novel reactions with hydrogen peroxide of histidine-containing nickel(I1)-oligopeptide complexes and nickel(I1)-induced structural changes in synthetic DNA. Biol. Trace Element Res. 21, 23-33. (210) Inoue, S., and Kawanishi, S. (1989) ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(I1) complex of glycylglycyl-L-histidine. Biochem. Biophys. Res. Commun. 159, 445-451. (211) Tofigh, S., and Frenkel, K. (1989) Effect of metals on nucleoside hydroperoxide, a product of ionizing radiation in DNA. Free Radical Biol. Med. 7, 131-143. (212) Cadet, J., and Teoule, R. (1975) Radiolyse gamma de la thymidine en solution aere6. I. Identification de hydroxyhydroperoxides. Bull. SOC.Chim. Fr. 3-4, 879-884. (213) Kasprzak, K. S., and Hernandez, L. (1989) Enhancement of hydroxylation and deglycosylation of 2’-deoxyguanosine by carcinogenic nickel compounds. Cancer Res. 49, 5964-5968. (214) Kensler, K. W., Egner, P. A., Taffe, P. G., and Trush, M. A. (1989) Role of free-radicals in tumor promotion and progression. In Skin Carcinogenesis: Mechanisms a n d Human Releuance (Slaga, T. J., Klein-Szanto, A. J. P., Boutwell, R. K., Stevenson, D. E., Sptizer, H. L., D’Motto, B., Eds.) pp 233-248, Alan R. Liss, New York.
The Role of Oxidative Damage in Metal Carcinogenicity Kazimierz S. Kasprzak* Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21 702 Received July 10, 1991
Introduction Several metals have been found to be carcinogenic to humans and/or animals (Tables I and 11), but the mechanisms involved in the process of tumor formation by these metals remain unclear. It is believed that transformation of cells, which may lead to cancer, results from a permanent (heritable) alteration in their genetic information introduced by a carcinogen. If this assumption is correct, any molecule that can bind with constituents of cell nuclei may affect the genetic code. From a purely chemical viewpoint, it is obvious that DNA, having an abundance of phosphate anions and nitrogen and oxygen donor groups, is an ideal binding partner for metal cations. Nuclear proteins bind metals as well. The structure and function of some of these proteins depend on the bound metal [e.g., “zinc fingers” (1, 2 ) , DNA polymerases ( 3 ) ] . Therefore, it is not surprising that exposure to toxic metals can result in their binding to the nucleus. A number of exogenous nonphysiological metals have been found to be associated with nuclear chromatin following an in vivo exposure ( 4 ) . Results like this must always be viewed with caution since the metals may become bound to the nuclear constituents during extraction. Nonetheless, precautions taken to avoid erratic results and elaborate verification experiments have provided convincing arguments that accumulation of many foreign metals in nuclear chromatin does really occur in vivo ( 4 ) . However, whether or not a metal ion other than the native DNA counterion, Mg(II), is capable of affecting the genetic code by merely binding to DNA and altering its conformation (5) remains an open question. Although several carcinogenic metals were tested *Address correspondence to this author at Building 538, Room 205, NCI-FCRDC, Frederick, MD 21702.
in vitro for their effect on DNA replication fidelity and found to decrease it (5-8), the concentrations needed to produce a significant number of replication errors in vitro would hardly be attainable in vivo without killing the cells. The electrovalent (ionic) and/or coordination bonds between metal cations and DNA are reversible (dissociable) and, as such, cannot produce all the lesions observed in nuclear chromatin of cells exposed to carcinogenic metal compounds. These DNA lesions, observed in both experimental animals and cultured cells (as discussed later), result from either breakage of existing covalent bonds or formation of new covalent bonds among organic molecules [e.g., DNA strand scission, depurination, DNA-protein and DNA interstrand cross-linking, and nucleobase modifications (8-11)]. Hence, not only the direct, mostly conformational effects of metal binding but also some other, obviously indirect effects of metals on nuclear chromatin must be considered. Interestingly, the plethora of lesions inflicted on nuclear chromatin by various carcinogenic metals can also be produced by oxygen radicals and/or other free-radical species generated, for example, by ionizing radiation (11-15). The similarity is striking enough to provoke a question whether or not the carcinogenic effects of metals are mediated by free radicals. An attempt to answer this question must deal with three major problems: (a) does the chemistry of carcinogenic metals allow for activation of oxygen and other species to produce free radicals under biologically relevant conditions, (b) do carcinogenic metals really mediate production of such radicals in living cells, (c) can the radicals avoid cellular scavengers and damage DNA? Answering the first question is relatively easy. All known carcinogenic metals [with perhaps one exception, Be(II)] have rich coordination and redox chemistry that may allow them to activate oxygen species under physio-
This article not subject to U S . Copyright. Published 1991 by the American Chemical Society
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Table I. Metal ExDosures Associated exposure/route metal Ni pyrometallurgy, hydrometallurgy, and electroplating of nickel/inhalation Cr chromate and chromate pigment production, chromium plating industries/inhalation As production and use of arsenic trioxide and its derivatives/inhalation, skin, and oral exposure
w i t h H u m a n Cancer" tumor location lung and sinonasal cancer
50
lung and sinonasal cancer
50
lung, skin and gastrointestinal cancers; precancerous dermal keratoses
7, 8, 56, 57
refs
"Limited evidence suggests that exposure to certain Fe, Cd, and Be derivatives increases risk of cancer in humans (7, 130, 131).
metal Be Cd Co Cr Fe Ni Pb Pt
Table 11. Metal Derivatives Producing Tumors in Experimental Animals" derivatives/exposure route tumor location BeO, BeHPO,/inhalation, iv, ios lung carcinomas, bone sarcomas CdIIacet, CdC12,CdS/sc, im, its local sarcomas, gonadal adenomas Coo, CoCl,, COS,CoO/im, sc, ios local sarcomas CaCrO,, PbCrO,/inhalation, sc, im lung carcinomas, local sarcomas iron(II1) dextran, Fe"'NTA/sc, im, ip local sarcomas, renal carcinomas NiO, Ni& cryst NiS, NiIIacet, Ni(CO),/inhalation, local sarcomas, lung and renal carcinomas sc, im, iv, iren, ioc Pb"acet/oral, sc, ip renal adenomas and carcinomas cis-Pt(II)/sc, ip lung adenomas, local sarcomas
refs 7, 130 7, 131 7, 132-134 7, 50 7, 88, 89, 134, 135 7, 50, 136 7, 134, 136-139 7, 134
" Also, aluminum(II1) dextran, manganese(I1) acetylacetonate, and titanium(1V) dicyclopentadiene induced local sarcomas after sc or im injections; NaAsOz given transplacentally and postnatally induced leukemias/lymphomas in mice; CuCI, and ZnSOl induced testicular tumors after local injection (7, 134, 138). Abbreviations: iv, intravenous; ip, intraperitoneal; sc, subcutaneous; im, intramuscular; ios, intraosseous: iren. intrarenal: ioc. intraocular: its. intratesticular: Coo, metallic cobalt powder; NiO, metallic nickel powder; acet, acetate; NTA, nitrilotriacetate. logical conditions (16, 17). Brief examples may include dioxygen (02) activation to superoxide (027during autoxidation of some metal aquo cations or hydrogen peroxide (H202)activation to hydroxyl radical ('OH) through the Fenton/Haber-Weiss chemistry (see below). Carcinogenicity of some metal compounds that produce tumors only when applied as crystalline powders may be due to heterogeneous redox catalysis depending on their surface properties (18-21). An unequivocal answer to question b is more difficult to provide because direct experimental data of the production of free radicals in vivo by carcinogenic metals are technically difficult to acquire. However, a positive answer to that question comes from indirect evidence based on identification of products of free-radical attack on biomolecules in metal-exposed cells. Presence of oxidatively modified DNA bases among such products provides an answer to question c. Some experimental data reveal that carcinogenic metals can inhibit cellular antioxidation systems and/or facilitate site-specific production of reactive species and thus sustain oxidative damage. In the present discussion, we shall review and evaluate the evidence substantiating a hypothesis of active oxygen involvement in the mechanisms of metal-induced carcinogenesis. First, we shall briefly consider the basic redox biochemistry of carcinogenic metals and the existing evidence on the type and magnitude of metal-induced redox damage to certain biomolecules. Then, we shall evaluate the relevance of that damage to carcinogenesis. Although various target organic molecules and carcinogenic metals will be considered, special attention in this review is given to DNA as a primary target for carcinogens and to two elements, nickel and chromium, that are well documented as carcinogenic to humans.
A Concise Redox Biochemistry of Carcinogenic Metal Derivatives A detailed presentation of the redox chemistry of carcinogenic metals is beyond the scope of this article. For the purpose of this discussion, let us focus on metal-mediated activation of oxygen species that ultimately results in the production of 'OH, the most powerful DNA-damaging radical, and/or related oxene metal derivatives.
The essentiality of metals for oxygen activation stems from Fenton/Habel-Weiss chemistry and/or autoxidation. The first allows for conversion of H 2 0 2and 02'-into the more reactive 'OH radical (22): Mn+ Hz02 M("+l)++ OH- + 'OH (1) M("+1)++ 02'- Ma+ + O2 (2)
+
-
---f
or
Mn+ + Oz'-
+ 2H+
-
+
M("+l)+ H 2 0 2
(3) Fenton-type reactions are driven by Cu(I), Fe(II), Co(II), and Ti(II1) aquo cations (22, 23). However, there are peculiar differences between Fe(I1) and Co(I1) reacting with H202in the nature of the radical products formed; besides the 'OH radical, Co(I1) also catalyzes production of 02'-(24). Combined reactions 2 and 3 which constitute a superoxide dismutase-like activity can be driven by aquo cations of Cu(I1) and Mn(I1) and by some complexes of Cu(II), Fe(II), and Ni(II), but not by the Ni(I1) aquo cation itself (25, 26). The major substrates for these reactions, H202and Oz'-, are generated in aerobic organisms during oxidative metabolism (27,28). The same oxygen derivatives may also be produced by some metal aquo cations or complexes directly from ambient oxygen, in the autoxidation reactions, e.g.: Fe2+ + O2 Fe3+ + 02'(4)
-
with subsequent production of H 2 0 2according to eq 3. Carcinogenic metal compounds interacting with H202may produce yet another active oxygen form, singlet oxygen '02 (29). Although it is the metal cation itself that drives electron transfer, the ligand environment in which the reacting metal is coordinated may have a profound effect on that transfer. Chelation of the cation may either enhance or inhibit the reactions. For example, autoxidation of Fe(I1) to Fe(II1) is enhanced by EDTA' and NTA (30), Abbreviations: ethylenediaminetetraacetic acid, EDTA; nitrilotriacetic acid, NTA; 2'-deoxyguanosine, dG; 8-hydroxy-2'-deoxyguanosine, 8-OH-dG; diethylenetriaminepentaacetic acid, DTPA; glycine, Gly; histidine, His; 8-hydroxyguanine,8-OH-G adenine, A cytosine, C; guanine, G; thymine, T; 8-hydroxyadenine, 8-OH-A; lauryl sulfate, sodium salt, SDS; thymidine, dT.
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Table 111. Reactivity of Ni(I1) Complexes with Peptides and Proteins toward Oxygen Species substrate active oxygen species produced/ligand degradation refs Ni(I1) complex with H202 'OH or Ni02+(?)/unknown 45, 46 Gbz, G~Y,,GlY,, GlY5 organic hydroperoxides, 02*-(?)/yes, mainly decarboxylation 45, 140 Gly,, Alal, Gly3NH2,Gly-Gly-His 02 46 'OH/unknown Gly-His H2Oz 26,47 'OH: 02'-,'02/yes, breakdown of the imidazole ring Gly-Gly-His H202 47 organic peroxides 'OH/yes, as above H202/unknown 26, 141 02.26, 47 'OH, 02'-/unknown Gly-Gly-His-Gly HzOz 26 H202/unknown 02.'OH/unknown 47 Ala-Gly-Gly-His, Gly-His-Ala H2Oz none 47 GIy2, Gly-Phe-Ala H202 142 unknown/yes, degradation to smaller peptides Va15-angiotensin-II-Asp1-,9-amide; polymyxin B H202 Ni02+ (?)/unknown 139, 141 human serum albumin H202 H202/unknown 02.26, 141
but inhibited by o-phenanthroline (31)and deferoxamine (desferal) (32). Ni(I1) in the presence of inorganic ligands is fully resistant to attack by O2 and/or H202. Oxidation of Ni(I1) and its reactivity toward H202may, however, be facilitated by chelation with several small peptides or proteins (Table 111). Likewise, Co(I1) aquo cations, which are stable under air, become sensitive to O2in the presence of chelators (24). Quite often, the organic ligand becomes the first target of oxygen activated by the coordinated metal cation, and the ligand degradation products may interact with other molecules, including proteins and DNA (33). In metal sulfides, one class of carcinogenic metal compounds, the inorganic ligand interacts with oxygen by itself. This may be responsible for the exceptionally high carcinogenic activity of some metal sulfides compared to other compounds of the same metals (34). For example, Ni3S2, one such highly carcinogenic sulfide, reacts with O2in vitro (35) and in vivo (36, 37) to produce the following Ni(I1) derivatives:
+ -
2Ni3S2+ O2 4NiS
4NiS
802
+ 2Ni0
4NiS0,
(5) (6)
Reaction 6 occurs in a stepwise fashion and goes through a NiS03 intermediate (37),which, besides being able to react further with 02,is also capable of inducing a unique, potentially mutagenic form of damage by deamination of DNA bases (38). The overall process of Ni3S2autoxidation apparently involves formation of active oxygen intermediates. As found in our laboratory, in the presence of oxygen, Ni3S2-catalyzedoxidation of dG to 8-OH-dG (39) and deaminatin of 5-methyl-2'-deoxycytidine to thymidine (38). Costa et al. (19)found that Ni3S2-richnickel mattes (to which nickel workers are exposed) catalyzed oxidation by O2of formate to C02'- radical. Oxygen-activating capacity was observed for particles of several other mineral sulfides, arsenides, and silicates (asbestos) (18, 20, 21). An important catalytic activity of carcinogenic transition metals is decomposition of organic peroxides (40, 41). Thus, alkyl hydroperoxides (e.g., lipid peroxides) are reduced to alkoxy1 radicals: ROOH
+ Mn+
-
RO'
+ OH- + M("+l)+
(7)
Likewise, the kinetics and proportion of decomposition products of nucleoside hydroperoxides, e.g., formed when DNA is irradiated in the presence of oxygen, depend on interaction with metal cations (42). The products include both stable and reactive derivatives and 'OH; the latter may further attack other DNA bases. For example, degradation of 5-(hydroperoxymethyl)-2'-deoxyuridine (a thymidine oxidation product) to 5-(hydroxymethyl)-2'deoxyuridine and 5-formyl-2'-deoxyuridineproceeds rapidly in the presence of Cu(I), Cu(II), Fe(II), and Sn(II), slower in the presence of Co(I1) and Ni(II), but not at all
in the presence of Fe(III), Mn(II), Mn(III), Al(III), Sn(IV), and Ca(I1) (42). Chelating agents, EDTA, DTPA, desferal, and Fe(II1)- or Cu(I1)-binding proteins either inhibit or enhance the degradation (42, 43). Owing to the high carcinogenic potency of nickel and chromium, the redox biochemistry of these two metals is worthy of more explicit discussion. At present, biological redox effects of Ni(I1) seem to depend on its ability to form an electron-transfer couple with Ni(II1) whereas the effects of Cr(V1) (chromate) depend on its reduction to lower oxidation states Cr(V), Cr(IV), and Cr(II1). Trivalent nickel was an exotic species with no apparent biological significance until 1971 when Paniago, Weatherburn, and Margerum discovered that the Ni(I1) complex with tetraglycine (Gly,) reacted spontaneously with 0, and produced an intermediate complex of Ni(II1) (reviewed in ref 44). Further studies showed that this reaction led to decomposition of the organic ligand with restoration of Ni(I1) so that the final result consisted of Ni(III/II)-catalyzed oxidative degradation of Gly, (44,45). It is now evident that Ni(I1) complexes with many other deprotonated peptides react with O2 in a similar way (Table 111). The kinetics of O2 reaction with the Ni(I1)-glycyl peptides diminishes in the order Gly, > Gly, > Gly, > Gly, > Gly-His (46). Also, Ni(I1) complexation with certain organic ligands causes this cation to react with HzOz according to Fenton-like chemistry (45).Ni(II)-Gly-Gly-His and Ni(I1)-Gly-Gly-His-Gly, besides converting H202into 'OH, also produce 02'-(46, 47). Formation of a highly reactive Ni-oxene radical (Ni02+)has been postulated by Nieboer et al. (26)for Ni(I1) bound to some His-containing peptides and human serum albumin. Such an oxene intermediate would be a powerful site-specific oxidizing agent. It is worth noting that neither Ni(I1) alone nor the His-containingpeptides alone exhibit any reactivity toward H202(26);likewise, complexes of Cu(II), Mn(II), Zn(II), and Cd(I1) are inactive (26). A significant result of the autoxidation reactions of Ni(I1) complexes, or HzOz disproportionation by these complexes, is the production not only of 'OH (or Ni02+)but also of other oxygen-, carbon-, and, perhaps, sulfur-centered radicals, which may attack biomolecules. Thus, autoxidation of the NinGly, complex leads to the formation of such intermediates as Gly,amide-N-methyl radical and its oxo and peroxo derivatives (45). Similarly, in the reaction of Ni(I1)-Gly-Gly-His complex with HzOz,the His residue decomposes to ammonia and aspartate (47),possibly through radical intermediates. These results indicate that Ni(I1) complexed with organic molecules renders these molecules vulnerable to attack by either O2or its more reactive derivatives, such as H202. Most recently, Ni(I1) was shown to facilitate Hz02attack on isolated DNA. The authors, Kawanishi et al. (48), assume that Ni(I1) binds to DNA and the complex formed reacts with H202. The final result is a strong site-specific
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cleavage of the DNA molecule by Hz02(presented later in more detail). Since the reaction cannot be stopped by the free ‘OH scavengers ethanol and mannitol, formation of a reactive intermediate nickel-oxygen complex is postulated at sites of Ni(I1)-DNA binding. Site-directing effects of Ni(I1) and Cu(I1) complexes with small DNAbinding proteins on DNA oxidation with H202are known (49). Chromium is well established as a carcinogen for both humans and animals but only in the form of the Cr(V1) compounds, Le., chromates and dichromates (50). The redox biochemistry of Cr(VI), relevant to carcinogenesis, is more complex than that of nickel. This chemistry has been studied in great detail by Wetterhahn et al. (e.g., refs 10 and 51 and this Forum) whose studies have led to formulation of an “uptake-reduction” model for Cr(V1) carcinogenesis (IO). Generally, under physiological conditions, Cr(V1) in the form of chromates is unreactive toward DNA. It is, however, capable of reacting with redox-active enzymes and small molecules to produce Cr(V), Cr(IV), and Cr(III), as well as oxygen- and sulfurcentered radicals. All these species can damage DNA. Hence, the ability of Cr(V1) to damage DNA depends on cellular redox systems. One such system depends on glutathione and produces Cr(V) and sulfur-centered glutathione-thiyl radical (10, 52) which may attack DNA. Cr(V1) may also be reduced by a variety of other cellular components, including molecules of the mitochondrial electron transport chain, carbohydrates, ascorbic acid, and H202(10,53-55). Reduction of Cr(V1) with H202leads to the production of ‘OH. Hydroxyl radical and Cr(V) are also generated when Cr(V1) is reduced enzymatically by glutathione reductase, a ubiquitous cellular enzyme (53). Further, Cr(V), the first intermediate of the reduction, has been recently found to decompose H202and produce ‘OH in a Fenton-type reaction (53). Therefore, Shi and Dalal (53,54)propose *OHas the “ultimate” carcinogenic species in Cr(V1) carcinogenesis. The molecular mechanisms whereby other metal carcinogens initiate and/or propagate oxidative damage (if any) are, thus far, only conjectural. Association between a particular metal’s carcinogenicity and a possible role of oxidative damage in its action may be anticipated if, for example, toxicity, mutagenicity, and/or carcinogenicity of this metal depends on its valency. This may be the case of arsenic. As(II1) compounds are much more toxic and carcinogenic than the As(V) derivatives (56,57). As(V) is reduced in vivo to As(II1). Hence, activation of As(V), like that of Cr(VI), also requires metabolic processing through redox reactions. There is no direct evidence, however, that As(II1) carcinogenicity is in fact mediated through oxygen activation. Thus far, some experimental data reveal that in vitro genotoxicity of As(II1) (as sodium arsenite) may be inhibited by superoxide dismutase and catalase, indicating increased production of H202and 02*in As(II1)-affected cells (58). The biochemistry of this phenomenon remains unknown. It might, perhaps, involve xanthine oxidase (discussed later). Involvement of active oxygen species can also be suspected in DNA damage by Cd(II), Hg(II), and Pb(I1). Thus, Ochi et al. (59) observed that DNA strand scissions in vitro by CdC12 could be prevented by superoxide dismutase or by an 02-free atmosphere. This indicated formation of 02*in Cd(I1)-affected cells. The authors speculate that the production of Q2’- is promoted by Cd(I1) through stimulation of some oxygen-activating enzymes (59, 60). Cantoni et al. (61) found that the addition of superoxide dismutase or catalase to HgClz-treated cells significantly reduced the extent of
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DNA single-strand breaks without reducing Hg(I1) uptake. Pb(I1) provides an interesting example whereby the ultimate oxidative cell damage is only remotely controlled by the primary insult, the Pb(I1) poisoning. Pb(I1) poisoning causes accumulation of 6-aminolevulinic acid (a heme precursor) which undergoes autoxidation accompanied by formation of reactive oxygen including H20z and 02’-. These radicals may then interact with cellular Fe(II)/Fe(111) to form ‘OH (62).
Genotoxic and Epigenetic Effects of Metal-Mediated Oxygen Activation. Chemical versus Biological Evidence It is of utmost importance for this discussion to acknowledge the fact that major substrates for metal carcinogen mediated oxygen activation, 02’-and Hz02,can be produced metabolically within mammalian cells, including the nucleus (63, 64). Hence, the substrates for further activation are present in the nucleus, and the carcinogenic metals may also get there. Also, the action of metal carcinogens triggers an inflammatory response which results in infiltration of the affected tissue by phagocytes with all their excretory oxidative weapons. Activated neutrophils are known to damage nucleobases in isolated DNA (65,66).Although it seems unlikely that all these products can diffuse among cells to finally reach nuclear DNA in the “target” cells (the cell population that gives rise to tumor), there is experimental evidence that at least one such product does, in fact, reach neighboring cells. Frenkel and Chrzan (67) incubated HeLa cells with stimulated polymorphonuclear leukocytes and found increased amounts of 5-(hydroxymethyl)-2’-deoxyuridine contents in DNA of the HeLa cells. Formation of this derivative could be prevented with catalase, indicating that H202was the diffusible intermediate responsible for oxidation. The authors speculate that, very likely, oxidation of d T and other nucleobases by Hz02was mediated by DNA-bound iron. Phagocytes are also known to cause bacterial cell mutations. Thus, His-requiring mutants of Salmonella typhimurium TAlOO were reverted into His independence following incubation with human blood leukocytes. Heat-killed leukocytes or leukocytes from a patient with chronic granulomatous disease (02*production defect) were inactive (68). It is worth noting that some carcinogenic metal sulfide particles were found to stimulate polymorphonuclear leukocytes to produce H20z (69). Most effective in this respect was Ni3S2followed by CdS and NiS2,whereas crystalline NiS and COSwere only marginally active. Noncarcinogenic BaS and MnS suppressed H202production. Surprisingly, soluble salts of Ni(II), Co(II), and Cd(I1) were ineffective [Ni(II), Cd(II)] or even suppressive [Co(II)] toward H202formation by the phagocytes. The magnitude of stimulation of the H202 formation by Ni3S2was a t the same level as that by a recognized tumor promoter, 12-0-tetradecanoylphorbol 13-acetate (69). Neither 02’nor H202is sufficiently active to react with biomolecules in the absence of metals (70-72);the most damaging oxygen radical is ‘OH (11, 73). Generally, the reactions of ‘OH with organic molecules, including DNA, can proceed without transition metals. Nonetheless, as discussed above, metals are essential for ‘OH production. They can also direct ‘OH attack on DNA to specific metal binding sites. It has been know from studies on the effects of ionizing radiation on biomolecules that ‘OH reacts with all constituents of nuclear chromatin. It can modify DNA bases and deoxyribose and produce DNA-protein crosslinks (11, 73). The pattern of chemical changes produced
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Table IV. Mediation of DNA Base Modifications by Carcinogenic Metal Derivatives in Vivo and in Vitro metal derivative experimental systemn result NiIIacet a single ip injection to Fischer rats elevated renal 8-OH-dG 16-48 h postinjection a single ip injection to BALB/c, B6C3F1, C3H, and elevated renal 8-OH-dG up to 48 h postinjection C57BL mice in BALB/c mice onlv exposure of NIH 3T3 and NRK-52 cells for 24 h variable (*25%) Ni(I1j concentration related and time-related changes in 8-OH-dG exposure of nuclear chromatin of human K562 cells to increase in DNA base products typical for 'OH NiCl,, CoC1, the metals plus H202 attack, including 8-hydroxypurines as above under air but without H202 increase in modified DNA bases, but to a lesser extent NiC12 than above incubation of calf thymus DNA with the dichromate NazCrzO, increased formation of 8-OH-dG plus GSH and H202 exposure of nuclear chromatin of murine S P - 2 / 0 cells FeCl,, CuSO, increase in DNA base products typical for 'OH to the metals plus Hz02 attack; chelation with EDTA or NTA suppressed Cu(I1) effect but enhanced Fe(II1) effect FeIIEDTA incubation of calf thymus DNA with Fe'IEDTA and increase in DNA base products typical for 'OH attack stimulated neutrophils FeII'EDTA incubation of calf thymus DNA or dG with FeII'EDTA increased formation of 8-OH-dG plus polyphenols and H,Oz FeCI,, Fe'IIEDTA incubation of calf thymus DNA with Fe(II1) and increase in DNA base products typical for 'OH attack; O,'--producing hypoxanthine/xanthine oxidase enhancement of this effect by EDTA system Fe'I'NTA a single ip injection to Wistar rats increase in renal 8-OH-dG up to 24 h postinjection Fe-rich asbestos incubation of calf thymus DNA with asbestos of various increase in 8-OH-dG in proportion to Fe contents types for up to 20 h
refs 78 93 93 75 75
10 76 65 143 71 79 144
Abbreviations: ip, intraperitoneal; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine;dG, 2'-deoxyguanosine; EDTA, ethylenediaminetetraacetate; GSH, reduced glutathione.
by 'OH in the base moiety of DNA is so characteristic that it can be used for identification of 'OH attack (13, 70, 73, 74). Such a pattern has been found most recently in DNA of nuclear chromatin isolated from cultured human K562 cells exposed to H,02 plus Ni(I1) or Co(I1) (75), or murine HyHEL-10 cells exposed to H202plus Cu(I1) or Fe(II1) (76). Most interestingly, unlike the other metals, Ni(I1) produced this effect even in the absence of H20z. This might be due to the ability of Ni(I1) complexes with certain peptide sequences, presumably present in the chromatin, to activate 02,as described previously (Table 111). The products of the metal-mediated H20zattack on DNA included the following (in the order of abundance): 8hydroxyguanine > 2,6-diamino-4-hydroxy-5-formamidopyrimidine > 8-hydroxyadenine > cytosine glycol > 4,6diamino-5-formamidopyrimidine> 5-hydroxy-5-methylhydantoin > 5-hydroxyhydantoin > thymine glycol > 5(hydroxymethy1)uracil 1 5,6-dihydroxycytosine L 2hydroxyadenine. It may be worthwhile to note that the relative abundance of the first three products listed is approximately 8:2:1 [for Ni(II), at the level of 30 nmol of 8-OH-G/mg of DNA]. Perhaps for this reason, the most easily detectable (in relative terms) and frequently analyzed products of oxygen radical attack on DNA in vivo and in vitro are the guanine derivatives, especially 8-OHdG. Data relevant to carcinogenic metal-mediated modifications of DNA bases are presented in Table IV. As can be seen in this table, elevated contents of the modified bases have been found thus far in DNA exposed to Fe(III), Ni(II), and Cr(V1) carcinogens and to asbestos. The importance of these findings to carcinogenesis relies on growing evidznce that some of these modifications, especially 8-OH-dG, constitute a potentially genotoxic DNA lesion. The 8-OH-dG lesion can be repaired (77), but judging by the development of tumors in animal tissues in which it has been detected (78, 79), the repair may be erratic. Several research groups determined the type of point mutation caused by the presence of 8-OH-dG in DNA templates (80-82). Despite initial controversies (compare ref 80 vs refs 81 and 82), it is now accepted that 8-OH-dG may code for A in addition to coding for C. This, in turn, leads to a G T transversion mutation (81,82).
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In our laboratory, this particular type of mutation was found to occur in the K-ras oncogene isolated from Ni3S2-inducedkidney tumors in rats (83). Codon 12 of this oncogene contained exclusively GGT GTT activating mutations. Both the development of tumors (shorter latency) and the incidence of codon 12 mutations of K-ras were greatly increased by the addition of metallic iron powder to Ni,S2. Since the iron powder did not produce any tumors by itself and did not affect the final tumor incidence by Ni3S2(83), the enhancement by iron of Ni3S2 carcinogenicity might be mediated through assistance in tumor progression. The K-ras mutation is not mandatory for nickel carcinogenesis since most of the tumors in the Ni3S2-onlygroup did not contain transforming mutations in K-ras. Instead, it may participate in tumor progression which is consistent with ras involvement in tumor latency (83). Besides 8-OH-dG, one product of thymidine oxidation, 5-(hydroxymethyl)-2'-deoxyuridine,is also potentially mutagenic; in contrast, thymidine glycols are not mutagenic (84). The mutagenic potential of most other 'OHmodified nucleobases is still debated or unexplored (14, 15). Research data available at this moment in abstracts only indicate that 8-OH-dG is likely to be more mutagenic by escaping from a proofreading function, whereas misincorporation at the 8-OH-dA lesion may be corrected (85). A more general mutation spectrum produced by agents generating oxygen radicals from 02,including Fe(II), Cu(I), and methylene blue plus light, was established by Loeb et al. in M13mp2 single-stranded DNA (86). The mutations were predominantly single-base substitutions which clustered characteristically for each agent. The most C frequent mutations were C T transitions and G transversions. The former type of mutation was not caused by deamination of C, and the latter was not caused by hydroxylation of G. It appears that the mutation type may depend not only on the given metal and/or oxygen radical but also on the target DNA. Thus, Loeb et al. (32) found Fe(I1) under aerobic conditions to be mutagenic also in ax174 am3 phage DNA. This time, the mutation involved mainly substitution of A for T opposite a template A (T A transversion), indicating that in this particular model
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iron directed the attack of the oxygen radicals on DNA predominantly at the adenine residues. Since depurination a t the A site has been ruled out, the authors suggest that a large fraction of mutagenesis by oxygen radicals consists of alteration in A, most likely a formation of 8-OH-A. Lack of the G T transversion mutation (see above) in both models is intriguing. Chemical models of Fe(I1)- or Fe(111)-mediated DNA oxidation (76,87) and in vivo experiments with FenlNTA (79)point at G rather than A as the base which is most sensitive to 'OH attack. The FemNTA complex appears to be a strong carcinogen to the rat and mouse kidney (88, 89),while iron(II1) chloride, Na'NTA, or A1"'NTA are not carcinogenic to the kidney or other tissues. Umemura et al. (79)found a significant increase of 8-OH-dG production in renal DNA of male Wistar rats given a single ip injection of FenlNTA, but not Na'NTA or iron(II1) chloride. Interestingly, Fe"'NTA, unlike Al"'NTA, also increases lipid peroxidation in the rat kidney (90).The cause(s) of differences relative to the exact sites of mutations by iron-directed DNA oxidation in various experimental models awaits (await) further elucidation. Nevertheless, besides Cr(V1) and Ni(II), Fe(II/III) has thus far provided the most convincing experimental evidence for long-suspected links between metal carcinogenesis and metal-mediated oxygen activation. In fact, owing to its relatively high redox activity (compared to other carcinogenic metals) and its tissue abundance, intracellular iron displaced by a toxic insult is often thought to be the ultimate metal carcinogen (28, 32,91). Copper, whose redox catalytic activity is even stronger, is, however, yet another candidate for this position (76). As shown by Kawanishi et al. (a), Ni(II) bound to DNA forms a complex that becomes capable of reacting with H202. The reaction cannot be stopped by free 'OH scavengers, indicating involvement of a reactive intermediate nickel-oxygen species at (or close to)specific Ni(I1) binding sites. The most vulnerable sites appear to be at the C, T, and G residues, and rarely a t the A residues. However, the location of sites of this cleavage predominantly at pyrimidine bases is somewhat surprising. Experiments on Ni reactions with individual nucleobases, nucleosides, and nucleotides a t physiological pH reveal generally weak bonding, preferably with the purine rings, mainly guanine, and no significant Ni(I1) interactions with the pyrimidine rings (92).The relatively strongest interaction with guanine is consistent with preferential oxidation of this base directed by Ni(I1) in vivo (78,79,93)and in vitro (75,93). At the DNA molecule, Ni(I1) is bound predominantly by the phosphate groups (94).The results of Kawanishi et al. (48)might, therefore, indicate that binding of Ni(I1) by the DNA molecule as a whole does not follow affinity rules established for separate nucleotides. Perhaps the difference results from the presence of other ligands [e.g., DTF'A (a)] which may form ternary complexes with Ni(I1) and DNA at DNA sites that can accommodate the other ligand. The carcinogen Cr(V1) does not significantly react with and damage DNA unless it is reduced metabolically to lower oxidative states. The intermediates of Cr(V1) reactions with small molecules and/or enzymes include Cr(V), Cr(IV), and Cr(III), as well as oxygen- and sulfurcentered radicals. All these species can damage DNA, though in different ways (10). The genotoxic effects include formation of DNA-chromium and DNA-radical adducts, DNA interstrand and intrastrand cross-links, DNA strand breaks, and especially DNA-protein crosslinks (53-55). For example, one such DNA adduct is
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formed with a glutathione-thiyl radical resulting from Cr(V1) reaction with glutathione (10, 52). Cysteine is cross-linked to DNA in the same way as glutathione (95). The DNA-protein cross-linking caused by Cr(V1) in CHO cells most frequently involves p95, p45, p53, and p54 proteins (95,96).Another product, 8-OH-dG, may result from Cr(V1) reaction with H202(10) or glutathione reductase (53).Besides nuclear DNA, an important target for Cr(V1) is also mitochondrial DNA (97,98). Mitochondria take up and reduce Cr(V1) to its most damaging form, Cr(V), which in turn has a much greater chance by virtue of proximity to attack the mitochondrial DNA than the nuclear DNA. A more general and potentially mutagenic effect of carcinogenic metals is depurination. In particular, Cr(V1) releases guanine, Cu(I1) releases adenine, and Ni(I1) probably releases adenine (the product was not definitely identified) from the DNA molecule (99).The effect is strongest with Cu(II), Cr(VI), and Ni(I1) and equivocal or not detectable with Pt(IV), Cr(III), Mn(II), Pb(II), As(V), Al(III), Zn(II), Ca(II), or Mg(I1). Schaaper et al. (99) speculate that the mechanism of depurination might have involved oxygen free radicals, analogously to DNA damage caused by Fe(I1) + H202(100).Significant enhancement by Ni(I1) of depurination of 2'-deoxyguanosine was observed in our laboratory (39)when dG was treated with the hydroxylating mixture of H202 + ascorbate (87)in the presence of Ni(I1). Involvement of active oxygen in this reaction was obvious. Interestingly, replication of DNA with an apurinic site may lead to the same G T transversion mutation as in the case of mutation caused by a 8-OH-G site (83). Carcinogenic transition metals promote lipid peroxidation (41).Although this effect cannot be considered as directly related to genotoxicity, it reflects biological oxidation-mediating capacity of a particular metal. Also, lipid peroxides may constitute a source of active oxygen species that, interacting with metals, will eventually cause genetic and/or epigenetic alterations. Enhancement of lipid peroxidation in rodents was observed for Cd(II), Co(II), Cu(II), Hg(II), Ni(II), Pb(II), Sn(II), and V(V) compounds. Metal toxicity and lipid peroxidation in rodents may be inhibited by administration of antioxidants, e.g., selenium or vitamin E (41).Most recently, Sugiyama et al. (101)showed that chromosomal aberrations and mutation caused by Cr(V1) in V79 cells were decreased in the presence of vitamin E.
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Effects of Metal Carcinogens on Cellular Oxygen-Handling Systems Besides being more or less directly involved in generation and activation of oxygen species, carcinogenic metal derivatives might also sustain oxidative damage to biomolecules indirectly, by inhibiting antioxidant cellular defense systems or enhancing oxygen activation systems (Table V). For example, in the liver and kidney of rats injected with Ni(II), a transient decrease in catalase and glutathione peroxidase activity during the first 24 h postinjection concurred with elevated lipid peroxidation (102). Catalase and glutathione peroxidase are also known to be inhibited by Ni(I1) in vitro (103,104). Superoxide dismutase appears to be less sensitive to Ni(I1) inhibition (102). However, great variability of the effects of Ni(I1) on catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, or superoxide dismutase in vivo, including not only inhibition but also enhancement, in different species, strains, and tissues, does not allow more general conclusions to be drawn as to the possible signif-
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Table V. Effects of Carcinogenic Metal Derivatives on Cellular Oxygen Activation and Deactivation Systems" metal derivative effect refs Ni"acet, NiClz inhibition of isolated CAT and GSH-Px, but enhancement of MPO in a Ni(I1) concentration 103, 104, 107 related manner transient inhibition of CAT activity in red blood cells, liver, and kidney of Ni(I1)-treated rats 102, 103 transient inhibition of CAT, GSH-Px, and GSSG-R in the kidney and liver, but not in muscle 102 of Ni(I1)-treated rats; no effect on SOD in kidney and liver, but decrease in skeletal muscle inhibition of CAT, SOD, and GSH in livers of four mouse strains, but variable effects on GSH-Px, 123, 124 GSSG-R, and GST in the same mice; no consistent patterns of Ni(I1) effect on the same enzymes in kidneys inhibition of hepatic GSH-Px activity, but increase in GSH, GSSG-R, and GST in Ni(I1)-treated rats 145, 146 depletion of hepatic GSH in 8-12 week old CBA mice but not in younger mice 147 Cd"ace t complete inhibition of SOD activity in bovine blood and in rat brain in vitro and strong inhibition in 105 various parts of the rat brain in vivo CdClZ possible stimulation of cell membrane bound pyridine nucleotide oxidase in human granulocytes and 60 rat alveolar macrophages P b"acet depletion of hepatic GSH-Px, SOD,and nonprotein SH groups and plasma vitamin E in mice treated 62 with Pb(I1) and bacterial endotoxin NaAsO, arsenite affects reactivity of reduced XO with O2by enhancing the 0, and/or 0,'- reactivity of the 106 reduced Mo center of the enzyme Abbreviations: acet, acetate; CAT, catalase; GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSSG-R, glutathione reductase;
GST, glutathione S-transferase; MPO,myeloperoxidase; SOD,superoxide dismutase; SH, sulfhydryl; XO, xanthine oxidase.
icance of antioxidant enzyme inhibition to oxidative damage by this metal (Table V). Unlike Ni(II), Cd(I1) appears to be a powerful and apparently specific inhibitor of superoxide dismutase, thus promoting metabolic 02' buildup in affected tissues (59,60,105). A t the same time, there are indications that Cd(I1) may enhance 02*production through stimulation of pyridine nucleotide oxidase (59,60). Likewise, arsenite has the potential to aggravate oxidation damage by R mechanism involving xanthine oxidase. Arsenite binds to the molybdenum center of xanthine oxidase (106). This binding enhances reactivity of the enzyme with O2 and makes it reactive with 0;-, one of its products. In effect, arsenite partially diverts the activity of xanthine oxidase toward production of relatively more Hz02than does As(II1)-free enzyme. Effects on oxygen-activating enzymes, or such enzymemimicking reactivity, can potentially increase oxidative damage by two more transition metals, Ni(I1) and Cu(I1). Ni(I1) was found to enhance myeloperoxidase (107), whereas Cu(I1) was found to mimic myeloperoxidase in a system with Hz02 plus NaCl (108). Both effects were observed in vitro, and it was not clear whether they could be reproduced in mammals or caused by other transition metals. In either case, the result consists of increased production of highly reactive hypohalite anion that may also contribute to the formation of lo2.The relevance of these effects to toxicity and especially genotoxicity of As(III), Ni(II), or Cu(I1) is, however, difficult to ascertain. Carcinogenic metal cations also have the potential to bind to and affect the reactivity of other cellular active oxygen scavengers such as glutathione, ascorbate, certain amino acids, fatty acids, and polyenols. In the case of F e W and N U ) , binding to ascorbate apparently increases oxidation of dG with HzOz(39). Likewise, Ni(I1) prevents glutathione and His from inhibiting dG oxidation (109). Moreover, His complexed with Ni(I1) becomes capable of enhancing dG oxidation with H202alone or H202+ asA similar effect was observed for Ni(I1) corbate and mannitol, a known free 'OH radical scavenger (109).
Conclusion Current hypotheses on the mechanisms of metal carcinogenesis stem from just one major phenomenon, i.e., the direct binding of metal cations to the constituents of cell
* K. S. Kasprzak and A. K. Datta, unpublished data.
nuclei (7,8). The binding is supposed to lead to the following effects, observed experimentally: (a) strand breakage and/or depurination of the DNA molecule, (b) DNA interstrand and DNA-protein cross-linking, (c) conformational changes of DNA with possible transition from B-DNA to Z-DNA, and (d) conformational changes of RNA and nuclear proteins including DNA polymerases. These effects may introduce both genetic and epigenetic alterations into target cells. The genetic alterations would result from mutations occurring during error-prone repair and/or replication of the damaged DNA (effects a and b); the errors may result from metal-affected template and/or metal-affected enzymes of DNA repair and replication (effect d). The epigenetic alterations would result from abnormal gene expression due to the exposure of segments of the genome that are normally repressed (effect c), or to erratic transcription and/or translation processes (effects b, c, and d). The latter may be especially associated with carcinogenic metal effects on gene regulatory proteins, the "zinc fingers" ( I , 2). Some other epigenetic effects of metals, e.g., inhibition of cell-cell communication (5,110, 111) and protein-protein cross-linking (33), are also possible. More details on the above are presented in recent reviews by Sunderman (5,7, 8). Experimental data accumulated thus far supports all the scenarios without, however, providing unequivocal clues as to the exact chemistry of the effects in question. There seems to be no doubt, at the present time, that carcinogenic metals do reach the cell nucleus and are retained there (1-4, 112). There remain, however, serious unanswered questions concerning the amounts of foreign metal that can be bound to the nucleus without arresting cell proliferation or killing the cell, the exact chemical form of the metal bound (aquo cation, organic complex cation, other complex form), and intranuclear distribution of the metal. Published data are insufficient to answer these questions. Nevertheless, general knowledge of cellular metal uptake, distribution, and toxicity indicates that the amounts are low, too low to produce serious conformational effects such as the B Z transformation of DNA and, perhaps, even too low to interact strongly enough with DNA to cause depurination and/or strand breakage. For example, DNA strand breakage in cultured cells was observed only for lethally high exposures to nickel carcinogens, but not for nontoxic exposures (113). A t nontoxic levels, metal binding to cell nuclei is limited not only by the gross cellular uptake capacity but also by abundance
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of competing nucleophiles (e.g., amino acids, peptides, proteins, fatty acids, etc.). The latter sequester the cations and thus decrease their availability for further interactions, e.g., with DNA (94). The most common nonlethal and potentially mutagenic effect of metals on nuclear chromatin observed in vitro and in vivo is DNA-protein cross-linking (lo,=,114,115).The strength of the cross-links may depend on the metal; nevertheless, most of the cross-links formed are relatively weak and dissociable in high ionic strength solutions, e.g., during SDS electrophoresis (116,117). Some of the cross-links, especially these produced by cis-Pt(I1) and Cr(III), are quite strong and can be disrupted only by strong metal chelators or reducing agents (95,114). Although the exact nature of these cross-links remains unknown, we may suspect that some of them are due to multivalent metal bridging (114).Some ternary metalDNA-protein complexes are, in fact, more stable than corresponding binary complexes (37,118).Other metalproduced DNA-protein cross-links are, however, much stronger; they are apparently caused by covalent bonds involving no metal bridging (119). DNA-protein crosslinking most likely results in chromosome aberrations in metal-treated cells. The most spectacular effects of this type are, perhaps, chromosomal aberrations observed in vitro and in vivo in a variety of cells including lymphocytes from nickel- and chromium-exposed workers (5). Interestingly, in Chinese hamster ovary cells, chromosomal damage by Ni(I1) is inflicted predominantly on the heterochromatic region of the X chromosome that is particularly rich in proteins (120). From the data presented above, it is easy to conclude that the variety and extent of nuclear chromatin damage by metals are greater than could be expected from mere binding of low amounts of metal cations. The type and magnitude of that damage is more consistent with growing experimental evidence that the metals affect nuclear chromatin not only through binding-related conformational distortions, but also through redox catalysis. The strongest support for this type of action comes from experiments in cell-free and in vitro systems. Relevant data were presented in previous chapters. They are quite strong and need no further comment, except perhaps for just one: DNA base modification studies by metals suffer from lack of experiments in cultured cells. Indeed, &(hydroxymethyl)-2'-deoxyuridine was identified in HeLa cells exposed to H202(from phagocytes), but not to a metal; involvement of iron in producing this derivative was only postulated by inference (67,121). Our own attempts to reveal the impact of Ni(1I) on &OH-dG production in NIH 3T3 and NRK-52 cells are still in progress; preliminary results indicate a rather weak and complex effect (93). The only direct in vivo evidence availabe thus far for oxidative damage to DNA bases by carcinogenic metal derivatives is limited to two papers (78,79) and one meeting abstract (93).In the papers, nickel(I1) acetate and Fe"NTA, both known to initiate renal cortical epithelial tumors by systemic injection, are described to increase renal 8-OH-dG contents within the first days after treatment. As stated before, 8-OH-dG in DNA causes point mutations consisting of G T transversions. Unfortunately, the tumors produced by nickel(I1) acetate or Fe'"NTA were not tested for oncogene activation. On the other hand, rat renal mesenchymal tumors induced with another nickel carcinogen, Ni3S2 (alone or plus metallic iron powder), were tested for oncogene activation, but the same kidneys had not been analyzed earlier for 8-OH-dG. The Ni3S2-inducedtumors were found to contain K-ras
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oncogene activated at codon 12 exclusively by the G T transversion mutation (GGT GTT) (83).Although the chemical and biological data suggest a causative association in the sequence nickel derivative increased 8-OH-dG K-rus mutation in the rat renal tumors, we still lack a direct confirmation that this is really true in a single experiment. Anyway, the association looks strong, especially if we consider one more fact: that renal mesenchymal tumors induced in rats with another carcinogen, methyl(methoxymethyl)nitrosamine,contained K-rus mutated by different base substitutions of codon 12 (122). It is noteworthy that the magnitude of Ni(I1) effects on 8-OHdG production in renal DNA may be species- and straindependent (78,93). No correlation has yet been made between this phenomenon and corresponding strain susceptibility of different strains of rats to renal carcinogenesis by Ni(I1). DNA depurination, another major effect of metals and oxygen radicals, has also been extensively studied in cell-free systems (see previous section). Obvious methodological limitations do not allow for obtaining direct evidence of this type of damage in surviving cells. So, we may only deduce that it really happens from the type of resulting mutations, if any. For example, the G T transversion mutation in renal tumors (83),originally linked to 8-OH-dG, could equally well be produced by depurination of the dG site. Depurination may occur concomitant with DNA strand scission, and both may result from *OH attack on the DNA sugar moiety (13). Modified sugars that remain in the DNA backbone form alkali-labile sites frequently observed in DNA from metal-treated cells (9,13,55). Thus, the presence of such sites may also indicate possible depurination. Formation of metal treatment related covalent DNAprotein cross-links in living cells is likely to involve *OH and other reactive oxygen intermediates (11,13,119). A good example of this possibility is cross-linking produced in isolated nuclear chromatin by H202 plus Fe"EDTA (119); H202alone does not cause the cross-linking. Further support for active oxygen involvement in the formation of DNA-protein cross-links comes from in vivo experiments in which this effect by Ni(I1) was greatly enhanced by Gly, and His: both known to complex Ni(I1) and render it reactive with O2and/or H202 (Table 111). Gly, also facilitated DNA-protein cross-linking by Ni(I1) in isolated calf thymus nucleohistone and protein-protein crosslinking among histones (33).Unfortunately, little is known about the influence of specific metals on the type and extent of the covalent cross-links and the exact chemistry of their formation. DNA-protein cross-links produced by 'OH generated by ionizing radiation in isolated calf thymus nucleohistone involve formation of covalent bonds mainly with the pyrimidine bases thymine and cytosine and the amino acids glycine, alanine, valine, leucine, isoleucine, threonine, lysine, and tyrosine (13).The significance of DNA-protein and possibly interprotein cross-linking in nuclear chromatin to somatic cell mutation and carcinogenesis remains to be defined. This damage can be repaired, though not completely (11). It is believed that persistent cross-links may impair functions of the nuclear matrix, especially during replication and transcription, and thus introduce genetic and epigenetic alterations into the affected cells (11). Besides the genetic and epigenetic effects resulting from direct metal interactions with the constituents of the cell nucleus, metals have the potential to aggravate overall
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oxidative stress by affecting cellular oxygen-handling systems and causing lipid peroxidation. A t present, the importance of these two phenomena to cell transformation and carcinogenesis is difficult to ascertain. From my own experience, it seems that both production of 8-OH-dG and lipid peroxidation by Ni(I1) in animals are highest in strains (e.g., BALB/c mice) which are low in glutathione and glutathione peroxidase compared to other strains (e.g., B6C3F1 or C3H mice) (93,123, 124). However, there are other indications that lipid peroxidation may not necessarily contribute to increased formation of 8-OH-dG (93). And finally, a delicate question: if oxygen activation plays a decisive role in metal carcinogenesis, why are the most biologically redox-active metals, copper and iron (76), not the strongest carcinogens? There are two possible answers to this question. The first answer must obviously consider bioavailability. Tight physiological control (chelation and compartmentalization) of these essential metals makes them inaccessible for adventitious reactions with oxygen species, e.g., H20z. Natural tissue ligands prevent iron and copper participation in radical reactions (125). Binding of iron by transferrin, lactoferrin, hemoglobin, myoglobin and storing it in the Fe(II1) form [less dangerous than the Fe(I1) form] and copper binding by albumin and ceruloplasmin greatly reduce the danger of uncontrolled redox reactions by these two metals (125). The carcinogenicity of the FemNTA and iron(II1) dextran complexes (Table 11) seems to be exceptional. It may reflect the ability of these complexes to protect Fe(II1) against its physiological ligands and, at the same time, their ability to deliver Fe(II1) to the target cells and sustain redox activity of Fe(II1). It seems possible that ligands like dextrane and NTA may also attenuate the inflammatory response to Fe(II1) and thus prevent the “overkill” effect, discussed below. There are not many iron-binding ligands that would provide all these functions at the same time and thus render this metal carcinogenic. Such ligands are unknown for copper. Generally, chelation of Cu(I1) inhibits its reactivity toward H202to a greater extent than chelation of Fe(I1) (13). In contrast, chelation of the Ni(I1) cation by proteins, peptides, and f or amino acids is essential for rendering it redox-active toward H202and/or O2 under physiologically relevant conditions (Table 111). The second answer to our question should consider an “overkill” effect. If it so happens that iron or copper escapes control (e.g., by parenteral administration), its powerful redox catalysis in conjunction with inflammatory response (active oxygen substrates) will result in killing, not just injuring, the target cells. I observed severe inflammatory/necrotizing effects of iron powder and iron(II1) chloride4in contrast to local cell immunosuppressive effects of Ni3S2in rat skeletal muscle (126, 127). The addition of mycobacterial antigen to Ni3S2,which greatly enhances phagocytic infiltration into the target tissue, prevents Ni3S2 carcinogenesis (128). Likewise, prevention of the immunosuppressive action of Ni(I1) by Mg(I1) inhibits Ni3S2 carcinogenesis (127). These findings obviously undermine a popular conviction of the possible villainous role of phagocytes in carcinogenesis, but the solution to this dilemma may also lay in the “overkill” approach: the phagocytes have been designed to destroy cells, not just tamper with their genetic code. The overall picture which emerges from our discussion portrays carcinogenic metal derivatives as multipotent reagents. They are capable of interacting with almost any cell constituent [including physiological metals (129)] to K. S. Kasprzak, unpublished data.
cause a plethora of more or less damaging effects in various molecules, including the mammalian genome. The effects may be direct, due to metal binding related conformational and functional distortions of biomolecules, or indirect, due to a variety of structural modifications of biomolecules by metal catalysis activated oxygen species. For uptake- and toxicity-related reasons, the catalytic effects of metals seem to be more important for carcinogenesis than the direct effects. Thus far, published data supporting the concept of the crucial role of oxidative damage in metal carcinogenesis is particularly strong for two of the most powerful human metal carcinogens, nickel and chromium. However, without excluding the contribution of other effects, oxidative damage seems to slowly be taking the leading role in explaining mechanisms of cancer causation and acute toxicity by other metals as well.
Acknowledgment. I am grateful to Drs. J. M. Rice and D. A. Wink for valuable critical comments on this paper and to Ms. K. Breeze for editorial help.
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