Chem. Res. Toxicol. 1991, 4, 592-604
592
Forum The Role of Oxidative Processes in Metal Carcinogenesis Catherine B. Klein,* Krystyna Frenkel, and Max Costa Institute of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received July 8, 1991
I ntroductlon Accumulating evidence demonstrates the ability of several carcinogenic metals to interact with and permanently alter the genetic components of living cells. The mechanisms of metal-induced carcinogenesis, however, are difficult to unravel since metal ion binding to critical cellular targets is weak and readily reversible. The study of metal carcinogenesis challenges our basic knowledge and understanding of mechanisms involved in DNA damage, mutagenesis, and tumor initiation, promotion, and progression leading to cancer development. In general, most metals that are carcinogenic in humans and animals (e.g., Ni, Cr, Cd, As, Be, Pb) exhibit low to moderate mutagenic activity in many assay systems (1). Although oxidative mechanisms have been increasingly implicated in metal mutagenesis, living cells and tissues are generally well protected from excessive oxidation products (2-7). Metals interact with all tissue and cellular components: membranes, organelles, proteins, metabolic enzymes, replicative and repair enzymes, nucleic acids, amino acids, and nucleotides. Therefore, it is difficult to investigate all metal/cellular interactions in relation to carcinogenesis, because the latter is a multistage process with potential for metal involvement at all stages. This review will briefly summarize emerging evidence which supports the notion that carcinogenic metals may be complete carcinogens, with the capacity to act as DNA-damaging, cell-transforming, and tumor-initiating/promoting agents via direct or indirect oxidative interactions. Figure 1depicts some of the mechanisms which may be involved in metal carcinogenesis as discussed in this review. Since oxidative mechanisms have not previously been highlighted in discussions of metal toxicology, this review will emphasize recent findings that suggest oxidative processes play an important role in metal carcinogenesis. Oxidative Processes Reactive oxygen species (ROS)' are necessary for the normal functioning of cells, and they participate in numerous oxidation-reduction processes (8-13). For example, oxidative phosphorylation is a major pathway of oxygen consumption in which O2is reduced by four electrons to water in the mitochondria (Scheme I). Normally, there is virtually no release of the partially reduced oxygen species from mitochondria, and those that escape are readily intercepted by cellular antioxidant defenses. Large amounts of ROS are also formed as bacteriocidal and tu-
* To whom reprint requests and correspondence should be addressed.
Scheme I. Reduction of Oxygena
,
[Fe2+,Cuc] , ,
e'
e' H202
[NADPH oxidase]
t
xanthine / xanthine oxidase
t I
LATI
I
'OH
-----)
H20
[pxl
RfoH H20
glucose I glucose oxidase a This simplified scheme depicts the sequential univalent reduction of molecular oxygen (0,) to water, which generates reactive hydrogen peroxide intermediates superoxide anion radicals (O;-), (H20z), and hydroxyl radicals ('OH). Some important catalytic enzymes shown include a few oxidases, catalase (CAT), peroxidase (Px), and superoxide dismutase (SOD).Also depicted is the nonenzymatic reduction of oxidized transition metal ions by 0,'-to their reduced forms, which then can catalyze reduction of H202 to 'OH.
moricidal agents by phagocytic cells, such as polymorphonuclear leukocytes (PMNs; neutrophils, granulocytes) and macrophages. Unfortunately, these important processes can be derailed from their usual pathways by a number of xenobiotics, which include some metal derivatives (13-28). Intracellular levels of oxidation products (ROS and other free radicals) can be increased by processes or insults that yield direct overproduction or deregulation of oxidants. Oxidant generation accompanies metabolism of mutagens/carcinogens involving Fenton-type reactions, perturbations of cytochrome electron-transfer pathways, membranemediated lipid peroxidation, radiation, introduction of direct-acting chemicals, and the inhibition of cellular antioxidant defenses. Once ROS escape from normal cellular controls, they have the potential to cause or contribute to a number of destructive processes and diseases, including cancer. To better understand their destructive role, we shall briefly review how ROS are formed, the genetic damage they cause, and how cells respond to this damage. Chemical Reactions. PMNs are normally activated by phagocytic stimuli such as bacteria and other opsonized particles. However, they can also be activated by nonphagocytic stimuli, including some allergens and tumor promoters (8, 10, 19, 24). Regardless of the stimulus, PMNs respond with an oxidative burst that is characterAbbreviations: PMNs, polymorphonuclear leukocytes (grmulocytea, neutrophils); ROS,reactive oxygen species; lo2, singlet oxygen; HOCl/ OC1-, hypochlorite; SOD,superoxide dismutase; TPA, 12-0-tetradecanoylphorbol 13-acetate (phorbol ester, PMA); 8-OHdG, 8-hydroxy-2'deoxyguanosine; GSH, glutathione (reduced form); AP, apurinic, apyrimidine sites.
0 1991 American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. 6,1991 593
Forum A
B
Chemotactic
. . PMN
y
.c
Chemotactic
C\W,
PMN
Figure 1. Metal-mediated oxidative processes in carcinogenesis. As depicted in part A, metals ( 0 )can generate reactive oxygen species (ROS) during the phagocytic uptake and dissolution of particulate metal derivatives (i.e., NiS) or during the intracellular reduction of metals (i.e., chromate). Some ROS,particularly H202,could pass into the nucleus t o react with DNA, and they have the potential to produce oxidized DNA bases and DNA strand breaks. In addition, metal ions ( 0 )themselves may directly alter the DNA. DNA-protein (s) interactions modified by metals can alter gene expression and initiate metal ion redox cycling (i.e., Ni2+/Ni3+)and ROS generation in direct proximity to the DNA. However, the Ni2+/Ni3+redox cycling is a proposed mechanism, which has not been as yet supported by actual measurements of paramagnetic Ni(II1) by electron spin resonance. Nickel binding occurs preferentially in heterochromatin, which makes up the inner lining of interphase nuclei. Part B depicts the potential tumor-promoting activity of metal derivative stimulated phagocytic cells (PMNs). Chemotactic factors produced by target cells and by PMNs will cause infiltration of numerous PMNs, which upon activation release more ROS and more potent chemotactic factors. This snowballing effect may be important in metal carcinogenesis, because metal exposure by inhalation frequently leads to lung accumulation of metal derivatives, followed by inflammation, and tumor development. Scheme 11. Formation of Reactive Oxygen Species by PMNs" n RNC19
-
+ [O,I
Activation by 1 Bacteria 21 Particles 3) Tumor Promoters
-To+ MPO
H202
a Polymorphonuclear leukocytes (PMNs)can be activated by and release an numerous stimuli to consume molecular oxygen (0,) oxidative burst of ROS which yields superoxide anions radicals (0;-) and hydrogen peroxide (H202). Hypochlorite (HOCl/OCl-) is enzymatically formed by myeloperoxidase (MPO)mediated oxidation of C1- by H202. Hypochlorite then reacts with amino groups of amino acids to generate mono- and dichloramines, a process which could lead to cell membrane disruption.
ized by the rapid consumption of molecular oxygen (0,) (Scheme 11). This oxygen is reduced to a superoxide anion radical (02*-)by an electron which is donated from NADPH, in a reaction catalyzed by membrane-bound NADPH oxidase. Although 0; - is relatively unreactive in aqueous media, it can inactivate some antioxidant enzymes, such as glutathione peroxidase and catalase (for review see ref 12). Moreover, it was shown to be a part of the chemotactic factor elaborated by cells treated with tumor promoters (29, 30). 02*is readily dismutated to hydrogen peroxide (H202)and O2 either spontaneously [with a rate constant of 8 X lo4 M-' s-l at pH 7.81 or enzymatically by superoxide dismutase (SOD) [with a rate constant of 2 X lo9 M-ls-l 3 (11). The nonenzymatic dismutation process of 02*-is pH-dependent and is highest a t its pK, of 4.8. The overall reaction can be written as 02'- + 02*--
2H+
H202 + 02
(dismutation reaction) (1) H202is the immediate precursor of the ultimate bacteriocidal and tumoricidal species, hypochlorite (HOC1/
OC1-), singlet oxygen (lo2),and the hydroxyl radical ('OH). Hypochlorite is formed enzymatically by myeloperoxidase-mediated oxidation of C1- ions by H202. It is a potent oxidizing agent (the main component of Clorox bleach) that readily interacts with amino and ammonium groups to generate another group of powerful oxidants, chloramines (8,31,32). This process causes disruption of cell membranes and release of membrane-derived oligopeptides. In the presence of excess H202,hypochlorite is reduced to C1- with concomitant generation of single oxygenH202 + C1-OC1-
+ H202
MPO
-
+ H2O C1- + H20 + 2'02 OC1-
(2)
(3)
H202can also be reduced to 'OH, one of the most potent oxidants known, by some transition metal ions (such as Fe and Cu) and their chelates (8, 11, 12): H202 Mn+ *OH OH- + M(n+l)+ (Fenton reaction) (4)
+
-
+
where Mn+denotes a reduced form of metal ion (i.e., Fe2+ and Cu'), and M(n+l)+denotes an oxidized metal ion (i.e., Fe3+ or Cu2+). This reaction is often referred to as the Fenton reaction. In cells, transition metals must be in a reduced form in order to reduce H202. The most common cellular reductant is 02* -. M(n+l)++ 02'- Ma+ + O2 (5)
-
The net result of reactions 4 and 5 is the so-called metal-catalyzed Haber-Weiss reaction: 02'-
metal + H202 catal~~st*OH + OH- + 02
(Haber-Weiss reaction) (6) During an oxidative burst, PMNs generate 02'-,which is a source of H202,and a reductant for metal ions. H202 is a source of hypochlorite, lo2,and 'OH. Of these ROS, only H202can traverse the plasma and nuclear membranes
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594 Chem. Res. Toxicol., Vol. 4 , No. 6, 1991 A
C
O
dR
dR
0
0
‘
R
I
dR
R
N
N
I
-1:) +-IN)
XN yCHzoH H2Nxk
0
R I
D
N\ t O H
I
dR
Figure 2. Some oxidized DNA base derivatives. (A and B) (+) and (-) cis-thymidine glycol, respectively; (C) 5-(hydroxymethyl)-2’-deoxyuridine;(D) 8-hydroxy-2’-deoxyguanosine.dR = 2’-deoxyribose. because it is neutral and unreactive unless it encounters a reductant of appropriate reducing potential. Upon reaching the nucleus, H,02 can cause site-specific DNA damage at locations that have bound transition metal ions ( 1 2 , 3 3 , 3 4 ) .Other ROS, such as ‘OH and hypochlorite, either are too reactive to migrate and usually act in the vicinity of their formation, or, like 02’-, require ion channels to enter cells. Products of Oxidative Processes in Vitro and in Vivo. Much of our historical knowledge regarding DNA and cellular modifications by ROS is derived from earlier studies of ionizing radiation. However, recent evidence indicates that similar modifications are caused by many xenobiotics, including metals. A brief review of these modifications will be presented here. DNA Base Modification. Potentially all the DNA bases can be oxidized in vitro by ionizing radiation (35-39). Qualitatively, the same types of oxidized DNA base derivatives can be formed by ROS generated by Fenton chemistry as by ionizing radiation (40,41). Over the last two decades, the most frequently studied oxidized bases have been thymidine glycol (19, 37, 41-48) and 5hydroxymethyl-2’-deoxyuridine(19,20,41,48-521, because the thymine moiety in DNA was thought to be the most susceptible to the modifying effects of ionizing radiation (53, 54). 8-Hydroxy-2’-deoxyguanosine(8-OHdG) is increasingly used as a marker of oxidative DNA damage because of its ease of detection by oxidative electrochemistry (55-61). However, one should bear in mind that in addition to these three types of oxidized DNA bases (see Figure 2) many others are also formed in DNA exposed to oxidative stress, as detected by gas chromatographymass spectrometry (35, 40). In recent years, formation of oxidized bases was demonstrated in DNA of cells exposed in vitro or in vivo to a variety of carcinogens. These include UV and y radiation, tumor promoters, polycyclic aromatic hydrocarbons, polyaromatic amines, nitro and nitroso derivatives, peroxisome proliferators, and salts of Fe, Cr, and Ni (19, 20, 34,41,46,47,49-51,55-57,62-67). Recent studies show the formation of 8-hydroxyguanine due to reactions of chromium with DNA in vitro (58) and in vivo (68). Rats treated with nickel acetate were shown to contain 8-OHdG in the DNA of the kidneys, which are target organs for Ni-induced carcinogenesis (59). Using gas chromatography-mass spectrometry analysis, a large number of modified DNA bases have also been detected in calf thymus DNA reacted with copper ions (69),and in the DNA from nickel-treated cultured human cells (70).
Po;
\ Figure 3. Generation of apurinic sites in DNA bases. Chemical attack at certain sites of DNA base (Le., N7-guanine) results in a change in electron density of the imidazole ring, which leads to the lability of the glycosylic bond between the base and the sugar. Spontaneous or alkali-induced disruption of this bond results in formation of an apurinic (AP)site. If not repaired, these AP sites may be subject to @-eliminationthat leads to strand breaks. Frank DNA strand breaks occur by direct disruptions of the sugar-phosphate backbone, resulting from ionizing radiation-induced or chemical-induced‘OH attack on carbons of the 2’-deoxyribose (54).
Apurinic Sites and DNA Strand Breaks. Numerous DNA-damaging agents induce abasic sites in DNA. Abasic sites are also referred to as AP (apurinic, apyrimidinc) sites, which are generated by cleavage of the glycosylic bond (Figure 3). In vitro studies have demonstrated the ability of Cr(VI), Ni(II), and Cu(I1) to depurinate bacteriophage 4x174 DNA (71, 72), leaving residual AP sites that are known to be premutagenic lesions (73, 74). Whereas Cu(II), Cr(III), Cr(VI), and Pt(1V) induce extensive lethal damage to the phage, only depurinated Cu(I1)- or Pt(1V)-treated single-stranded DNA was shown to undergo mutation when the DNA was transfected into SOS-induced bacterial spheroplasts (72). AP sites exhibit a distinct pattern of mutagenesis with A preferentially inserted opposite the lesion (73). This pattern of A insertion is typical for mutagenesis by noncoding lesions. Mutagenesis by AP sites in Escherichia coli requires SOS processing (75, 76). Oxidative mutagens including ionizing radiation, hydrogen peroxide, bleomycin, neocarzinosta.tin, and copper(1)1,lO-phenanthroline complex have also been shown to create abasic sites by oxidation of the sugar moiety (for review see ref 77). It has been suggested that these oxidized AP sites in double-stranded DNA are less stable to chemical cleavage at neutral pH than other AP sites, but may also be more resistant to enzymatic repair (77). DNA damage and repair can be evaluated by alkaline elution, which detects DNA strand breakage. DNA strand breaks frequently occur by chemical or radical attack on the sugar moiety of the DNA backbone (53). Direct DNA strand breaks by metals have been examined in both human and hamster cell DNA (for review see ref 78). Other techniques such as nucleoid sedimentation analysis and the DNA unwinding assay have also been used to determine the extent of metal-induced DNA strand breakage (79). Using these methods, it has been suggested that DNA strand breaks are not crucial DNA lesions for metal-induced cytotoxicity. Evidence of DNA strand breaks produced by specific metals will be discussed in more detail later in this review. Mutagenicity of Oxidant-MediatedProcesses. ROS generated by carcinogens are mutagenic, transform cells,
Forum
and are carcinogenic (80-86). In particular, chronic inflammation (during which high levels of ROS are produced by PMNs and macrophages) is known to contribute to many types of cancer (for review see ref 86). The ROS generated by phagocytic cells cause formation of oxidized and are muDNA bases in coincubated cells (51,87,88) tagenic in bacteria and mammalian cells (81-83,89,90). Some of the oxidized base derivatives themselves (Le., 5-hydroxymethyl-2’-deoxyuridineand 8-OHdG) are mutagenic (91-95),as are thymidine hydroperoxides2(96-98). Since ROS (generated enzymatically or by ionizing radiation) as well as the products of ROS interactions with DNA bases are mutagenic, it is logical to conclude that at least some types of oxidative DNA damage also lead to mutations. Repair of Oxidative Modifications. DNA repair is relevant to metal carcinogenesis because there is accumulating evidence for the repair of oxidative DNA damage (4,37, 79,99,100). It is clear from recent studies that oxidized DNA bases can be removed from prokaryotic and eukaryotic DNA by both base excision and nucleotide excision repair pathways. Some important enzymes involved in base excision repair include the recently described E. coli endonuclease that removes %hydroxyguanine from double-stranded DNA (101,102), as well as previously described enzymes such as 54hydroxymethy1)uracil glycosylase in mammalian cells (103,104), 5-(hydroxymethy1)cytosine glycosylase (105), thymine glycol glycosylase (106,107),E.coli endonuclease I11 (39), and the more general redoxyendonuclease(99).Recently, the bacterial UUFABCnucleotide excision repair complex has been demonstrated to be active against thymine glycol and some apurinic sites (108, 109). However, metal ions also inhibit enzymes (including repair enzymes) directly or indirectly via the formation of oxidation products (for review see ref 100). Inducible responses for coping with excessive oxidative imbalances such as that mediated by oxyR and soxR in bacteria are reviewed elsewhere ( 4 ) , and a potentially similar adaptive response has been described for human lymphocytes treated with hydrogen peroxide prior to X-irradiation (110). The generally held view that oxidative base modification constitutes a detrimental DNA lesions that is repaired or induces mutation may not be the only interpretation. Oxidized bases may also represent a normal DNA modification akin to cytosine methylation, or oxidation that occurs at methylated cytosines may result in removal of the oxidized cytosine with concommitant loss of cytosine methylation at that site. Although experimental support for this idea is not yet forthcoming, it seems clear that oxidized bases have the potential to cause profound differences in protein binding to nucleic acids, which could detrimentally alter gene expression.
Genotoxic Consequences of Oxidative Reactions The ensuing discussion will review some recently accumulated data which are supportive of oxidative mechanisms in the carcinogenic action of certain metals. Although less detailed than the accompanying articles by Standeven and Wetterhahn (111)and Kasprzak (112),our discussion will revolve around chromium, nickel, and some other metal compounds for which there is now significant indication of active oxidative mechanisms. This review
* U. Pate1 and K. Frenkel, Mechanism of mutagenicity by 5-(hydroperoxymethyl)-2’-deoxyuridine,an intermediate product of ionizing radiation. Manuscript in preparation.
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is not intended to be exhaustive, but rather aims to outline an emerging picture in which redox mechanisms and radical chemistry furnish certain metal compounds with the capacity to act as complete carcinogens. In general, the more relevant experimental studies will be those that have identified oxidation products or redox chemistry in metal-exposed cells and tissues. More circumstantial evidence is provided by experiments in which modulation of metal-induced genotoxic end points can be shown by a wide variety of oxidation inhibitors or potentiators. Chromium. The mutagenicity and carcinogenicity of chromium has been the topic of recent extensive reviews (78,113,114),and only the most salient features will be quoted here. Experimental evidence indisputably depicta chromium as a genotoxic agent in most in vitro and in vivo assays. These tests include assays that detect DNA strand breaks (115, 116),alkali-labile DNA sites (71,721,chrosister chromatid exchanges mosomal aberrations (117,118), (119), DNA-DNA or DNA-protein cross-links (for review see ref 78), and mutations in both bacterial and mammalian cell systems (120-122).Although these data have been mainly acquired from experiments utilizing Cr(V1) compounds, there is some indication that other oxidation states of chromium, Cr(V), Cr(V) complexes, or Cr(III), are also genotoxic. A significant discussion has arisen regarding which chromium valence state is the ultimate carcinogen in vivo. An understanding of chromium chemistry and bioavailability is essential to our interpretation of experimental results (78). It is now well-documented that chromium in its hexavalent state is proficiently taken up by living cells via passive anion transport channels (123) and is reduced intracellularly through the lower valences to Cr(II1) (113)(see Figure 1). Cr(II1) accumulates in cells due to its inability to diffuse through the cellular membrane (113,124-126).Although the relative cellular uptake reof Cr(V1) > Cr(II1) was previously reported (78,127), cent atomic absorption spectroscopy data show both intracellular and nuclear accumulation of chromium ions in sodium chromate treated Chinese hamster V79 cells within 3 h of exposure (128). The significance of these findings is that reduced forms of Cr ions accumulate in direct proximity to cellular DNA. Particularly active is Cr(V), which is produced by reduction of Cr(V1) by microsomes, mitochondria, HzOz,NADPH, ascorbate, glutathione (58, 121, 129),glutathione reductase (130),and vitamin B2 (131). Cr(V) is also formed in vivo in chick embryo red blood cells (68). Cr(V1) has been shown to form 8hydroxyguanine in vitro (58) and to induce both intrastrand DNA and DNA-protein cross-links in vivo (68, 132-135). Another reduced Cr ion, Cr(III),has been shown to bind to isolated nuclei (133),to interact with nucleotides and nucleic acids (137,138), and to interact in vitro with DNA to promote decreased fidelity of DNA replication by polymerases (139,140). Although these effects are not usually thought to be due to oxidative processes, it has recently been suggested that Cr(II1) can undergo redox cycling in the presence of biological reductants (141,142). These effects of Cr(II1) may be due to ion pair complexes. Chromate reduction in the presence of Hz02can produce 02’and ‘OH in vitro (129). As discussed earlier, these radicals have the potential to cleave DNA (11-13). In several recent studies, the DNA-damaging ability of chromium derivatives was found to occur by mechanisms that may be defined as oxidative. Participation of ROS in the generation of Cr(V1)-mediated DNA damage in cultured human cells was suggested by demonstrating that this damage is inhibited by mannitol, SOD, and catalase
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(79). Single-strand breaks in supercoiled PM2 DNA produced in vitro by reduction of chromate by glutathione (143) were found to be specifically inhibited by 'OH scavengers such as DMSO, formate, and benzoate, and by the H202scavenger catalase (144). Since strand breaks were not inhibited by SOD (144, 145), it is believed that 02'does not readily generate 'OH here, but rather serves as a source of H202. Similar results were obtained for the in vitro inhibition of DNA strand break formation induced by Cr(V)-glutathione octahydrate (an intermediate of GHS-mediated chromate reduction) (144), suggesting a complicated reduction mechanism whereby glutathionyl radicals and reduced chromate cause formation of H202 and 'OH (145). In the work of Kortenkamp et al. (I&), FeC13 (1-10 pM) was required for induction of strand breaks by both Cr(V1) and a Cr(V)-GSH complex. However, at Fe concentrations greater than 10 pM, the DNA damage was independent of Cr and likely due to the iron alone. These results are consistent with those of Aiyar et al. (58) who found that chromate and GSH do not cleave pBR322 DNA in vitro if trace iron is removed. These results point to the importance of endogenous metals (such as iron), and the Fenton reaction, in the potentiation of effects induced by carcinogenic metals. Chromosomal gaps and breaks are the predominant aberrations produced in cells treated with chromate (119). It has been shown that chromate-induced aberrations are random in the genome (117) and are produced throughout the cell cycle with a slight S-phase preference for DNA strand breaks (135). Vitamin E, an antioxidant which is known to be protective against oxidative damage (7), including that induced by ionizing radiation (146), has recently been shown to reduce the frequency of chromosome aberrations and mutations produced by sodium chromate in Chinese hamster V79 cells (147). Previously, a similar diminution of DNA strand breaks, of inhibition of glutathione reductase, and of cellular levels of Cr(V) was shown in cultured mammalian cells (148, 149). Genetic end points such as cytotoxicity and mutagenicity have also been demonstrated to occur by mechanisms which include, but are not restricted to, oxidative processes. Numerous investigators have shown chromium compounds to be mutagenic in bacterial assays (reviewed in ref 120), including several of the classical Salmonella tester strains such as TA98 and TAlOO (150). Recently, Cr(V1) and Cr(II1) were tested for their mutagenicity in the Salmonella tester strains, TA102 and TA2638, which are sensitive to oxidative agents (142). Both strains are sensitive to chromium mutagenesis by hexavalent and trivalent compounds; however, Cr(II1) mutagenesis for both strains is dependent upon aerobic growth conditions, whereas Cr(V1) mutagenicity is dependent on the presence of oxygen in TA102 but not TA2638. One conclusion is that chromium compounds are multimechanistic in their genotoxic actions. Another is that oxygen and ROS contribute to some of the DNA-damaging reactions. The antioxidant ascorbic acid (vitamin C) decreases the mutagenicity of Cr(VI) in Salmonella (122),which may be due to extracellular reduction of Cr(V1) to Cr(III), thereby reducing chromium uptake by the cells. Many chromium compounds are mutagenic in several systems (114). However, chromium(II1) is not overtly mutagenic in most systems, presumably because of its inefficient uptake by living cells (151). Vitamin E was recently reported to inhibit the mutagenicity of sodium chromate at the hypoxanthine phosphoribosyltransferase locus in Chinese hamster V79 cells (147), and it was pre-
viously noted that the same antioxidant could reduce chromate cytotoxicity in these cells (149). Vitamin B2has the ability to reduce Cr(V1) in the presence of H202,and to enhance DNA strand breakage while at the same time reducing cytotoxicity to V79 cells (149, 152). However, it is unknown if chromate mutagenicity at other mammalian mutagenesis loci is inhibited by antioxidants. From our own investigations of chromate mutagenicity in V79 cells, and in radiosensitive V79-derived transgenic G12 cells carrying the bacterial guanine phosphoribosyltransferase gene (153), it is known that the mutagenic response to chromate (and other metals including Ni and Hg) is highly dependent upon accurately defining a nonlethal mutagenic dose range for the metal. The effective dose range is frequently narrow and difficult to delineate, due to an apparent metal-induced detachment of cells from the culture plates, which complicates toxicity estimates that are based on relative cloning or plating efficiencie~.~ Although the debate continues as to which chromium metabolic intermediate or metal complex may be the ultimate mutagenlcarcinogen, several studies point toward Cr(V)- or Cr(V)-glutathione (GSH) complexes as the species of interest. Although the Cr(V)-glutathione (GSH) complex may be the ultimate DNA-damaging agent, this species is short-lived in solution and is rapidly reduced to the less reactive Cr(II1)-GSH (144). In the process, chromate reduction provides ample opportunity for the intracellular production of ROS and other radical species, which themselves are capable of interacting with proteins, nucleic acids, and subcellular organelles (i.e., mitochondria; 154,155). Thus, it is likely that chromium induces DNA damage by direct reaction with a reduced chromium species, or by ROS generated as a result of chromate reduction. Nickel. Nickel is a well-documented human and animal carcinogen especially in its particulate forms ( I , 78, 156-158, but demonstration of its mutagenicity has been elusive. As recently reviewed (159) and supported by new experimental data (160),nickel compounds generally are not mutagenic in bacterial assays, including the oxidation-sensitive Salmonella strain TA102 (161). In cultured mammalian cells, NiC1, has been shown to exert a weak mutagenic response a t the hypoxanthine phosphoribosyltransferase locus in Chinese hamster V79 cells (162) but not in C3H mouse cells (163). It is weakly active at the thymidine kinase locus of L5178Y mouse lymphoma cells (164). In addition, nickel is synergistically mutagenic with UV (165), alkylating agents, and some bulky carcinogens (reviewed in refs 157 and 159). Although some nickel particulates such as crystalline nickel subsulfide (aNi3S2)are nonmutagenic in V79 cells (160),crystalline NiS has recently been shown to be quite mutagenic (166) at the bacterial guanine phosphoribosyltransferase locus of the transgenic V79-derived G12 cell line (153). These transgenic cells are hypersensitive to mutagenesis by Xrays and bleomycin (167),suggesting their potential to detect mutagenesis mediated by oxidative processes. Alternatively, this cell line may have the capacity to detect mutations such as deletions, which may not be recoverable at the hypoxanthine phosphoribosyltransferase locus on the V79 X chromosome. Although the bioavailability of nickel compounds is very different from that of chromate, it is nevertheless an essential determinant of nickel genotoxicity and carcinogenicity. Insoluble nickel compounds such as nickel sulfide and nickel subsulfide are among the most carcinogenic C. B. Klein, and E. T. Snow, unpublished results.
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Forum nickel compounds. As recently reviewed (158),the bioavailability of nickel is dependent primarily upon particle size, charge, and the efficiency of phagocytic uptake of the particulate nickel compounds (see Figure 1). Electron microscopy of crystalline nickel subsulfide treated hamster cells provided evidence that these particles do not enter the cell nucleus but rather aggregate around the nuclear membrane (168). However, more recent ultrastructural analysis shows the penetration of some minute nickel particles into the nucleus of human lymphocytes but not Chinese hamster V79 cells (160). The nuclear uptake of soluble nickel ions from the particles is likely to be due to dissolution of nickel via acidification of the membrane-associated vacuoles containing the particles. The recovery of these nuclear Ni ions in TCA precipitates suggests their association with macromolecules such as DNA (169). The genotoxicity of nickel can be ascertained by the ability of assorted nickel compounds to produce DNA strand breaks and DNA-protein cross-links in vitro (for review see refs 157 and 159), and by their ability to induce chromosomal aberrations ( 1 70, 171) and sister chromatid exchanges (165,172)in cultured human and rodent cells. Vitamin E inhibition of chromosomal aberrations induced by insoluble nickel sulfide, but not the soluble nickel chloride, has been demonstrated in Chinese hamster ovary cells (173),implying that the phagocytic process by which the nickel enters the cell is capable of generating ROS which may then react with cellular DNA. This study also supports the concept that phagocytosis of the insoluble nickel compounds allows for higher intracellular concentrations of the potentially reactive Ni(II), since the more soluble ionic nickel compounds (which are less carcinogenic) are not as readily taken up by mammalian cells. Experimental evidence suggests that nickel is primarily a clastogen, which induces chromosomal alterations in cultured hamster cells with preferential activity toward heterochromatin ( 1 71, 174). Heterochromatin is densely packaged DNA, which is often associated with transcriptionally inactive regions of the genome. The propensity for nickel interaction with heterochromatin was confirmed in a study of Chinese hamster ovary versus mouse (C3HlOT1/2) cells (117) which contain nearly equal amounts of heterochromatin concentrated in different genomic regions. It was subsequently found to be even more specific for the induction of decondensation and deletions in the heterochromatic long arm of the hamster X chromosome (175). Our laboratory has recently described the capacity of the intact hamster X chromosome to restore dominant cellular senescence to an immortal, nickel-transformed, male Chinese hamster cell line which has a large chromosomal deletion in the heterochromatic region of the X chromosome (176). Senescence has been demonstrated with the transfer of both hamster and human X chromosomes into nickel-transformed Chinese hamster cell lines, but not with transfer into spontaneously transformed hamster cell lines such as CHO and V79 (177). Interestingly, senescence was also induced by the X chromosome in a nickel-transformed male Chinese hamster cell line which did not exhibit microscopically visible deletion of the X chromosome (176). Restoration of senescence is important to our basic understanding of nickel carcinogenesis,since senescence implies negative regulation of normal cell growth and is akin to the negative modulation of tumor suppressors on tumor cell growth. It is possible that the preference for nickel interactions with the heterochromatin is related to the affinity of nickel for these protein- and Mg(I1)-rich chromatin regions.
Although Ni(I1) is not highly reactive with DNA, it can potentially undergo redox cycles between Ni(III)/Ni(II) valence states when Ni(I1) is complexed to proteins (178, 179). However, to our knowledge electron spin resonance studies demonstrating formation of Ni(II1) in these reactions have not been carried out. Trace contamination by oxidatively active metals (i.e., Fe, Cu) may be sufficient to produce oxygen radical formation in any of these reactions. Evidence for nickel interactions with the nuclear matrix includes the recovery of nickel cross-links with actin and other matrix proteins, as reviewed in detail by Coogan et al. (157). Such interactions could inappropriately turn off or delete active genes. Alternatively, Ni-protein interactions and resulting redox reactions could occur on the periphery of the nucleus with effects on regulation of transcription. The inopportune binding or release of regulatory proteins from transcription factor recognition sites could result in alterations of gene expression, which may be relevant to nickel carcinogenesis but may be unrelated to direct DNA modification. These Ni-protein interactions or redox effects need not be abundant to be extremely significant with regard to their site-specific action. Several other diverse lines of evidence support a role for ROS metabolism in the carcinogenicity of nickel. Historically, the dependence on molecular oxygen was found for the solubilization of nickel subsulfide in rat serum in vitro, an effect which could be inhibited by NADPH and enhanced by albumin or amino acids (180). Hepatotoxicity following exposure to nickel chloride results from lipid peroxidation (reviewed in ref 157), in which the involvement of 'OH is implied by the antagonism of this peroxidation by EDTA and benzoate (181). In addition, the presence of 11 different oxidized DNA-base residues demonstrated in the chromatin of nickel-exposed cultured human cells proves the involvement of 'OH in their formation, particularly since the yield of these adducts was reduced by EDTA and nitrilotriacetate and enhanced by SOD. Since none of these studies have measured the formation of Ni(III), it is difficult to ascertain the mechanism for the oxidative damage. SOD was previously shown to stimulate DNA damage by Cu(I1) or Fe(II1) and (40). Other Metals. Mercury compounds such as mercuric chloride and methylmercury chloride are extremely toxic to cultured cells (182, 183). Although organic mercury compounds were shown to be weakly mutagenic in Chinese hamster V79 cells (184),our recent mutagenesis data with methylmercury were not significant in either V79 or in the derivative transgenic cell lines4 (153). Mercury produces DNA strand breaks with kinetics that are similar to strand break induction by X-rays (185, 186). Recently, Costa et al. reviewed (187)the dose-dependent production of SODor GSH-inhibitable mercuric chloride induced DNA strand breaks in cultured human fibroblasts, which supports previous data obtained using cultured hamster cells (182, 185). Methylmercury chloride induced ROS, which can be mitigated by deferoxamine, have recently been identified in rat brain tissue (reviewed in ref 187). Ochi et al. (189)reported the participation of ROS in the production of cadmium chloride induced DNA strand breaks in cultured mammalian cells. More recently, cadmium sulfide was shown to elicit Hz02formation by human polymorphonuclear lymphocytes (26),an effect that may be correlated with inflammation and tumor promotion as
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discussed in more detail below. Using cultured human fibroblasts, Snyder (79) reported the ROS-mediated production of DNA strand breaks by Cd, Mg, Mn, Cr(VI), Zn, and selenite. Although 'OH scavengers were effective in reducing the breaks induced by the divalent Cd, Mg, and Mn ions, they were much less effective with Cr(VI), Zn(II), and selenite. SOD inhibited the breaks caused by Mg, Cr, and Zn, whereas catalase inhibited all breaks except those formed by Mn and selenite. As mentioned above, GSH enhanced the DNA strand breaks produced by Cr(V1). Among these metals, however, only Cr and Cd are well-established carcinogens, although it is also known that metals can often act synergistically in carcinogenesis (190). Rossman and collaborators (191)previously reported the capacity of several metal compounds to induce bacteriophage h in the microscreen assay, which is a function of the SOS response induced by DNA damage. These results have recently been reevaluated in consideration of the available data base for Salmonella mutagenesis and rodent carcinogenesis (192). It is interesting to note that, among the tested carcinogenic metals, beryllium and cadmium chlorides did not cause either h induction or Salmonella mutagenesis whereas lead nitrate, nickel acetate, and manganous chloride induced h prophage, but were not mutagenic. Only potassium chromate was positive in both genotoxicity assays. These tested metal compounds were predominantly water-soluble. It is unknown, however, if reactive metal complexes formed in the cells, similar to those created by the intracellular reduction of Cr(V1) (123-125). For some metals, it may be the insoluble compounds which are more carcinogenic. Both iron and copper can participate in Fenton reactions. These reactions may yield biological damage including mutations (8, 9, 11-13, 193). Recently, Fe- and Cu-modified 4x174 DNA was transfected into SOS-induced bacterial spheroplasts and scored for mutations (194). The Fe(I1)-modified DNA was mutagenic by mechanisms that suggested the activity of ROS (194),and the mutagenic spectrum of the DNA sequence alterations was nonrandom (195). Similar findings have been reported for copper [both (Cu(1) and Cu(II)],with evidence of sequence-specific hot-spots and a high incidence of C T transitions (196). Iron and copper, as well as other ubiquitous endogenous metals (e.g., Mg, Zn), however, are not believed to be highly carcinogenic, although it is possible that a higher incidence of cancer may be associated with human diseases related to excessive metal accumulation (196,197). Examples of some of these metabolic disorders are Wilson's and Menkes's disease, which are related to accumulated copper (197), and idiopathic hemochromatosis from accumulated iron (197, 198).
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Cellular Transformation by Carcinogenic Metals Cellular transformation is characterized in part by altered cell growth, anchorage-independent proliferation, loss of contact inhibition, and immortality, which may result from perturbations of intracellular communication, activation of oncogenes, or the loss of tumor suppressor genes. Landolph (199) tabulated recently available data on metal responses in transformation studies and correlated these data with mutagenesis in mammalian cells. In another study, Miki and collaborators (200) reported that nickel disrupts cell-cell communication by oxidative mechanisms similar to those induced by TPA (12-0-tetradecanoylphorbol 13-acetate). However, Mikalsen (201) did not detect interference with the formation and maintenance of normal cellular communication channels with several
metal salts including Ni, Cd, Pb, Cr(VI), and Cr(II1). Whether oxidative processes play a role in metal-induced transformation has not been explored in detail. Chromium compounds primarily induce human cancers from respiratory exposures leading to lung carcinomas and, to a lesser extent, nasal and pharyngeal carcinomas (114, 202). Gastrointestinal cancer is infrequent, and skin cancer has not been noted even in cases of severe skin exposure. In human and animal studies, the least soluble Cr(V1) compounds are the most carcinogenic, and human exposure to Cr(II1) alone has never been reported to be carcinogenic. Chromium tumors arise primarily at exposure sites in accordance with the uptake-reduction model (113) for cellular chromate metabolism, since sublethal chromium can be trapped within exposed cells where it may initiate DNA damage. Exposure site carcinogenesis is also in accord with the potential for chromium to elicit inflammatory/ tumor promotional responses, as discussed below. The reduced potential for tumor development a t locations distant from the exposure site is in agreement with evidence that extracellular reduction of Cr(V1) by the antioxidants in body fluids is effective in preventing cellular uptake of chromium, since Cr(II1) does not enter cells (114,122). The imbalance between localized defenses and tumor-initiating/promoting events is essential in all carcinogenesis, including that induced by metals. Although the exact circumstances surrounding nickel carcinogenesis are different from those for chromate (157, 159), several similarities can be pointed out. One of the most important common features is that nickel tumors are also primarily located in the regions which sustain predominant exposures, mainly respiratory sites (203). Evidence for both DNA-damaging and tumor-promoting activities by certain nickel compounds already exists (26,59).
Tumor Promotion as an Oxidative Function of Carcinogenic Metals Activation of PMNs by tumor promoters such as the phorbol ester TPA leads to production of H20zthat can migrate into other cells and cause DNA damage. Oxidized DNA bases are often used as markers of this damage (24, 34, 47, 51, 52, 57, 61). Recently, it was found that even in vivo TPA mediates generation of H202(48,204)and the production of oxidized bases in mouse epidermal DNA (48). In addition to stimulating phagocytic cells, tumorpromoting agents also induce oxidative activation of cultured cells, such as HeLa and mouse epidermal cells (51, 204, 205). The oxidative activation is characterized by formation of ROS and oxidative DNA damage, as measured by H202,DNA strand breaks, and oxidation of DNA bases, all of which are substantially enhanced when PMNs are also present. We recently found that carcinogenic insoluble sulfides of Cd, Ni (26), and CaCr0: stimulate human PMNs in vitro and induce production of 02'-and HzOzin a manner similar to that caused by the tumor promoter TPA. Ni and Cd sulfides also activate rat (Fisher 344) PMNs in the absence, and even more so in the presence, of autologous plasma.6 In vitro exposure of CdS also stimulates production of H20zby macrophages of rainbow trout elicited in vivo with bacteria prior to collection of the macrophages.' In vivo exposure of trout to CdClz results in macrophage populations that generate much more 02* K. Frenkel, unpublished data. Z. Zhong, W. Troll, and K. Frenkel, unpublished data. N. A. Enane, J. T.Zelikoff, K. S. Squibb, and K. Frenkel, unpublished data.
Forum and HzOzin response to TPA activation than cells isolated from the nonexposed fish.8 Hence, all of these metal salts can induce formation of DNA-damaging ROS that are characteristic of tumor promoters. Ni3Sz., NiS, and CdS are known to accumulate and persist in the lungs, a target tissue for their toxicity and carcinogenicity (157, 159, 206). These salts induce formation of chemotactic factors that cause infiltration of phagocytes (207, 208) and then, as we found, can also stimulate those cells to generate damaging ROS (26). The ensuing inflammation may lead to further damaging consequences. For example, human lung cells intermittently exposed to ROS generated by TPA-stimulated human PMNs were transformed within 4-5 weeks. When injected into nude mice, these transformed cells gave rise to tumors that were metastatic (85). These cells were also analyzed for the presence of oxidized DNA bases using the 3Hpostlabeling technique (41). Levels of both 5-(hydroxymethyl)-2’-deoxyuridine and 8-OHdG gradually increased in the lung cells with exposure to activated PMNs and were 10- and 60-fold higher, respectively, after 5 weeks? H202 is the most likely mediator of oxidative DNA modification in those lung cells because it can migrate through the membranes almost as easily as water while other ROS cannot (8, 12). When it reaches the nucleus, H20zcan cause oxidation of DNA bases at sites containing bound transition metal ions. In most cases it will likely be Fe ions chelated by DNA. However, if other metal ions that can act as reductants [such as Cr(V)] are bound to the cellular DNA, they may reduce H202and cause formation of ‘OH (or ‘OH-like species) that will oxidize the DNA bases. That this may be the case is shown by induction of strand breaks in the DNA of V79 cells incubated with chromate in the presence of vitamin B2,which reduces it to Cr(V), and by a Cr(V)-mediated increase in the generation of ‘OH (131). Moreover, strand breaks as well as 8-OHdG are formed in DNA exposed to Cr(V1) in the presence of H202(58,129). Although by itself Ni(I1) cannot reduce H202,when it is a part of a chelate (i.e., complexed to histidine-containing oligopeptide), it acts like SOD with 0;-and reacts with H202as well (209,210). PMNs stimulated by NiS produce and H202,whereas those stimulated by Ni3S2 both OZ*apparently generate only H20z(26). As yet, it is not known whether these two Ni salts activate PMNs by different Soluble pathways or if Ni3S2catalyzes dismutation of 02*-. Ni(I1) and Co(I1) salts gradually decompose 5-(hydroperoxymethyl)-2’-deoxyuridine(211), which is known to be formed in DNA through the action of ionizing radiation as are other nucleoside hydroperoxides (54,96, 97,212). Thus, the presence of these metal ions may change the profile of oxidative DNA damage. It was shown recently (59) that initiation with nickel acetate and promotion with phenobarbital cause in vivo formation of 8-OHdG (a product of ROS attack) in rat kidney DNA, which is also a site of Ni-induced carcinogenesis. Although Ni acetate is water-soluble, the kidney is known to accumulate such salts (172) and even 2 days after exposure a substantial amount of Ni is still present there. The same group also showed that H202,in the presence of Ni,S2, hydroxylates 2‘-deoxyguanosine to &OHdG (213),which strengthens the hypothesis that H20z is an intermediate necessary for metal-mediated oxidation of bases in DNA. Hence, similarly to the organic tumor promoters, carcinogenic metal
* J. T. Zelikoff, K. S. Squibb, D. Bower, and K. Frenkel, unpublished data. H. Wei, D. Corvese, A. B. Weitberg, and K. Frenkel, unpublished data.
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salts induce formation of oxidants and oxidative DNA damage, processes thought to be important in tumor promotion/progression (24,26,214).
Conclusions and Future Directions Cumulatively, the data presented in this review suggest that carcinogenic metal derivatives can induce oxidative DNA modifications, which are implicated in many aspects of tumor initiation, promotion, and/or progression. By acting directly on cells or indirectly through stimulation of phagocytes, metals can induce the production of ROS, which in turn can mediate the oxidation of bases in the DNA of target cells. Additionally, the metals may promote the development of tumors by maintaining inflammation in the region of the metal concentration, and inflammation has been known to act as a cocarcinogen (86). Hence, the prooxidant activity of certain metals may be the basis for their capacity to act as complete carcinogens, as exemplified by tumor production close to the initial exposure sites. In vitro studies of metal-mediated DNA damage have been extremely informative, and it is now well established that numerous carcinogenic metal salts can elicit these deleterious responses. It is also clear that antagonism/ potentiation can alter metal-induced genotoxicity. In the future, the detailed search for markers of oxidative processes in important transformation/ carcinogenesis genes may offer evidence that inappropriate oxidation has occurred. Equally important will be studies on modulation of gene expression by metal-mediated nucleic acid-protein interactions or redox processes, which can occur either within or outside the nucleus. Finally, it will be interesting to determine whether a functional role for nucleic acid oxidation exists.
Acknowledgment. We thank Drs. Elizabeth T. Snow, Sofia Cosentino, Xin W. Wang, and Jerome J. Solomon for their discussions and critical comments regarding the manuscript. In addition, our appreciation is extended to Jane Galvin, Judy Battista, and Susan Benninghoff for their excellent secretarial assistance. This work was supported by Grants CA 37858 and CA 49798 from the National Cancer Institute and ES 04895, ES 04715, and ES 05512 from the National Institute of Environmental Health Sciences.
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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
