Metal carcinogenesis: mechanistic implications.

Pharmac. Ther.Vol. 53, pp. 31-65, 1992 Printed in Great Britain. All rightsreserved

0163-7258/92$15.00 © 1992PergamonPressLtd

Associate Editor: D. GRUNBERGER

METAL CARCINOGENESIS: MECHANISTIC IMPLICATIONS ELIZABETH T. S N o w Nelson Institute of Environmental Medicine, New York University Medical Center, Long Meadow Road, Tuxedo, N Y 10987, U.S.A. Abstract--Cancer epidemiology has identified several metal compounds as human carcinogens. Recent evidence suggests that carcinogenic metals induce genotoxicity in a multiplicity of ways, either alone or by enhancing the effects of other agents. This review summarizes current information on the genotoxicity of arsenic, chromium, nickel, beryllium and cadmium compounds and their possible roles in carcinogenesis. Each of these metals is distinct in its primary modes of action; yet there are several mechanisms induced by more than one metal, including: the induction of cellular immunity and oxidative stress, the inhibition of DNA metabolism and repair and the formation of DNA- and/or protein-crosslinks.

CONTENTS 1. Introduction 2. Mechanisms of Metal Carcinogenesis: An Overview 2.1. Initiation: Mutagenesis and altered gene expression 2.2. Promotion: Oxidation and signal transduction 2.3. Progression: Self stimulation by tumor cells 2.4. Metastasis: Tumor cell migration 2.5. Transformation in vitro: Oncogenes and gene expression 3. The Epidemiology of Metal Carcinogenesis: Mechanistic Implications 3.1. Arsenic: Threshold effects and multiple targets 3.2. Chromium: Speciation and complex chemistry 3.3. Nickel: Uptake and cellular metabolism 3.4. Other metals: Diversity of action 3.4.1. Beryllium 3.4.2. Cadmium 4. Mechanisms of Genotoxicity and Carcinogenesis by Specific Metals 4.1. Arsenic: DNA repair and gene expression 4.1.1. Bioavailability and uptake 4.1.2. Inhibition of DNA repair processes and mutagenesis 4.1.3. Chromosomal effects 4.1.4. Gene amplification and expression 4.2. Chromium: Redox processing and DNA interactions 4.2.1. Bioavailability and uptake 4.2.2. In vitro cell transformation 4.2.3. DNA and chromosomal damage 4.2.4. Bacterial and mammalian cell mutagenesis 4.2.5. Mechanisms of chromium mutagenesis 4.2.6. The role of chromium(III) 4.2.7. Metabolic modulation of chromium genotoxicity 4.3. Nickel: Protein interactions and cellular responses 4.3.1. Uptake and bioavailability 4.3.2. Cellular transformation and mutagenesis 4.3.3. Chromosomal and DNA damage 4.3.4. The role of oxidative processes 4.3.5. Interactions with heterochromatin and magnesium 4.4. Other metals: Protein inhibition and DNA repair 4.4.1. Beryllium: Cellular immunity and nucleic acid metabolism 4.4.2. Cadmium: Protein interactions and gene expression 5. Summary Acknowledgements References 31

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E.T. SNOW 1. INTRODUCTION

The evidence for cancer induction by occupational or iatrogenic exposure to compounds of metals such as arsenic, beryllium, chromium and nickel is convincing and well documented. These metal compounds are among several dozen environmental agents, including tobacco products, asbestos and ionizing and solar radiation, for which there is sufficient evidence for human carcinogenicity (IARC, 1980, 1987, 1990). Other metals such as cadmium, lead and mercury are also suspected human carcinogens. The mechanisms of cancer induction by these compounds are not well understood. In most cases it is not known whether the metal compounds act as tumor initiators or tumor promoters, or both. Some metal compounds such as chromium (Wolf et al., 1989), nickel (Ciccarelli and Wetterhahn, 1985) and cadmium (Koizumi and Waalkes, 1990) can interact directly with nucleic acids and promote genotoxic damage or mutagenesis. Some metal compounds may also alter gene expression (Chin et al., 1990; Cosma et al., 1991; Hamilton and Wetterhahn, 1989). They may act alone or in synergy with other agents. Nickel (Schubert et al., 1987; Smialowicz et al., 1987) and chromium (Kumar and Barthwal, 1991) species stimulate cellular immune responses, while nickel and cadmium uptake can promote the release of active oxygen species (Zhong et al., 1990). Experimental evidence for elucidating mechanisms of metal carcinogenesis is abundant yet often conflicting. Because carcinogenesis is in itself a complex process and metals are a diverse group of compounds, there is unlikely to be one unified mechanism for metal carcinogenesis. In fact, each metal compound is likely to contribute to human carcinogenesis by multiple mechanisms. However, a careful examination of the evidence may allow us to discern several key processes for each metal that are consistent with its potential role as a tumorigenic agent. This review will outline current evidence related to molecular mechanisms of genotoxicity and alterations in gene expression and DNA repair induced by several well established metal carcinogens. The primary focus of the review will be on those metals for which there is the best evidence of human carcinogenesis and for which there is the most information relating to genotoxic mechanisms: arsenic, chromium and nickel. Other probable metal carcinogens, such as beryllium and cadmium will be discussed briefly. The mechanistic implications of the data will be discussed with respect to the role of metals in human carcinogenesis.

2. MECHANISMS OF METAL CARCINOGENESIS: AN OVERVIEW Four overlapping stages of carcinogenesis will be considered: initiation, promotion, progression and metastasis. Metal ions may be involved at each and all stages of the carcinogenic process. Figure 1 summarizes these stages and the potential involvement of different metals at each stage. Morphological transformation of cells in culture will also be discussed as an experimental system which is a strong predictor of carcinogenic potential. In vitro cell transformation can be considered to include aspects of both tumor initiation and promotion, depending on the assay conditions and the cells used. 2.1. INITIATION:MUTAGENESISAND ALTERED GENE EXPRESSION

Carcinogenic metal compounds have been shown to produce both bacterial and mammalian cell mutagenesis in vitro. Chromate salts, in particular, tend to give positive responses in most short term genotoxicity assays (De Flora et al., 1990). Although metal compounds are frequently not strong mutagens, several carcinogenic metal compounds produce more than additive mutagenesis when applied with a second mutagen (Christie, 1989; Dubins and Lavelle, 1986; Rossman et al., 1992). Unlike many organic carcinogens, metal compounds do not produce known types of direct mutagenic DNA damage or adducts. They can however produce general oxidative DNA damage (Rodriguez et aL, 1991; Standeven and Wetterhahn, 1991a) or DNA crosslinks which may occur directly or as a byproduct of metal metabolism within a cell (Wedrychowski et al., 1985). Oxidative DNA damage induced by redox cycling of specific metal ions in the presence of H202 has been shown to be mutagenic in bacterial cells (McBride et al., 1991; Tkeshelashvili et al., 1991). However,

Metal carcinogenesis: mechanistic implications

33

Nonnel Cell ~ DNA Demlme~

DNA Repair

INITIATION

or,

UV/ AIk/ X-rays

AlteredDNA Replication Cr(lll),NI / Be

. / METASTASIS

lb

PROMOTION AlteredGene ImmuneResponse Expression & NI, Cd, Be Cd, As, Cr, NI ?

L

Mint or

Altered Ca / Mg Homeostasis

PROGRESSION FIG. 1. The role of metals in carcinogenesis.Carcinogenesis is a multistage process, involving initiation, promotion, progression and metastasis. Metal compounds can be involved at many different stages of carcinogenesisand each type of metal is likely to be involved at more than one step and via different mechanisms.

the actual mutagenic lesions produced by these processes are not yet known. It is also unknown whether carcinogenic metals which are capable of redox cycling in vitro (e.g. chromium, nickel, cadmium) can induce the production of active oxygen species by phagocytic cells in vivo (i.e. nickel and cadmium) (Zhong et al., 1990) can also produce mutagenic DNA damage in target tissue cells in vivo.

Different metals may act in more than one way to initiate tumorigenesis in vivo. Cancer initiation may not require the production of classic point mutations. Although some oncogenes such as cHa-ras can be activated by specific base substitution mutations (Bos, 1989; Kumar et al., 1990), tumor supressor genes or anti-oncogenes may be inactivated by either point mutations (Hollstein et al., 1991) or by deletion mutagenesis (Kaden et al., 1989; Sager, 1988; Solomon et al., 1991; Weinberg, 1991). Tumor initiation (or progression) can also occur via transcriptional activation, recombination, or amplification of other oncogenes such as myc and erbB2 (HER-2/neu) (Schwab, 1990). These mutagenic events may or may not require the production of specific DNA lesions. Recombination and deletion mutagenesis, for example, can result from the formation of DNA strand breaks (Ashby et al., 1990), however, they may also result from the inhibition of DNA replication (Huang et al., 1989; Preston, 1982) or repair (Chung et al., 1991; Koberle and Speit, 1991). Carcinogenic metal compounds of chromium, nickel, arsenic and cadmium have all been shown to induce various types of DNA recombination and/or amplification in mammalian cells in vitro.

2.2. PROMOTION: OXIDATION AND SIGNAL TRANSDUCTION

Metal salts can promote altered gene expression (Flamigni et al., 1989) or modify intra- or inter-cellular communication (Mikalsen, 1990). These and other alterations in signal transduction are generally considered to be related to tumor promotion rather than initiation. Tumor promotion is also associated with the production of oxidative bursts by phagocytic cells and cellular recruitment. Phagocytosis of particulate metal compounds has been shown to promote these processes in vitro (Zhong et al., 1990) and in vivo (Knight et al., 1991; Shirali et aL, 1991; Sunderman et aL, 1989; Wiernik et al., 1983).

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E.T. Srqow 2.3. PROGRESSION:SELF STIMULATIONBY TUMOR CELLS

Tumor progression can be studied independently of promotion by examining the characteristics of a developing tumor and the cells within it. Tumor promotion can be thought of as a series of events that lead to tumor progression. Progression is associated with altered tumor cell metabolism, the acquisition of additional phenotypic characteristics and multiple genotypic changes (Kaden et al., 1989; Lehman et aL, 1991; Loeb, 1991). As cells within the tumor progress they may acquire the ability to produce increased amounts of active oxygen species (Szatrowski and Nathan, 1991). These active oxygen species, in turn, may induce additional somatic mutations within both the tumor cells and adjacent normal cells. These processes may also be facilitated by changes in the oxidative metabolism of the cells and by changes in immune status of the host. Carcinogenic metals such as nickel (Judde et al., 1987; Smialowicz et al., 1987) and chromium (Kumar and Barthwal, 1991) can interfere with the redox status of mammalian cells in vitro and have strong effects on the cellular immune system (De Flora and Wetterhahn, 1989; Rodriguez et al., 1991; Shainkinkestenbaum et al., 1991; Standeven and Wetterhahn, 1991a). Having already achieved an altered metabolic profile, those tumor cells with increased growth potential, a more rapid growth rate, or any other advantage, will undergo selection such that the characteristics of the cells within the tumor undergo gradual change. 2.4. METASTASIS: TUMOR CELL MIGRATION

The final stage of malignant tumorigenesis is metastasis. This occurs when the cells within the tumor mass acquire the ability to travel and colonize at sites distant from the initial cancer. Metastatic potential is associated with relaxed intercellular communication, altered cellular membranes and enhanced proliferative capacity. One potential role for exogenous metals in this process may be related to alterations in calcium regulation. Calcium plays an essential role in intraand inter-cellular communication and interference with calcium binding or regulation may be pivotal in both tumor progression and metastatic potential (Whitfield, 1990). 2.5. TRANSFORMATIONI N

VITRO:

ONCOGENESAND GENE EXPRESSION

Morphological transformation in vitro is an experimental endpoint which has been associated with changes in oncogene activation and alterations in signal transduction (Landolph, 1990). All known carcinogenic metals have been shown to induce morphological transformation of cells in culture in one or more assay system (Biedermann and Landolph, 1987; DiPaolo and Casto, 1979; Landolph, 1990). Even metal salts which have not been shown to be mutagenic to mammalian cells in vitro, such as chromium(III) chloride (Biedermann and Landolph, 1990) and sodium arsenite (Biedermann and Landolph, 1987), have been positive in cell transformation assays. It is likely that cellular transformation or anchorage independence may occur by mechanisms such as alterations in gene expression or signal transduction that are independent of the gene mutation and inactivation processes which are typically assayed in mammalian cell mutagenesis protocols. It is intriguing that different valence forms of the same metal, such as chromium(VI) and chromium(III), may be differentially active in one or both of these assays. These findings suggest that uptake and metabolic processing of certain metal salts may give rise to different genotoxic endpoints and that cell transformation (a more general endpoint) can occur by different processes than mammalian cell mutagenesis. Cell transformation in vitro displays characteristics of both carcinogenic initiation and promotion.

3. EPIDEMIOLOGY OF METAL CARCINOGENESIS: MECHANISTIC IMPLICATIONS 3.1. ARSENIC: THRESHOLDEFFECTS AND MULTIPLE TARGETS Humans have been exposed to arsenic for centuries. Compounds of arsenic have been used as poisons and as pharmaceuticals. Arsenic is also a recognized human carcinogen (Chen and Wang, 1990; Lronard, 1984; Pershagen, 1981; Pinto et al., 1978; Stohrer, 1991). In a recent follow-up study on a large population of Taiwanese exposed to arsenic in their well water, it was shown that arsenic


35

ingestion is associated with increased risk for cancers of the skin, lung, fiver,bladder, kidney and prostate (Chen and Wang, 1990). Occupational exposure to arsenic compounds is also associated with skin and lung cancer (Gilman and Swierenga, 1985). Unlike other carcinogens for which there is limited quantitative information on exposure dose-response parameters, arsenic carcinogenicity shows clear evidence for a threshold below which there isno response (Stohrer, 1991). Furthermore, unlike most other human carcinogens, arsenic is not carcinogenic in rodent species (Gilman and Swierenga, 1985). Although arsenic compounds have been shown to increase the carcinogenic response of rats to benzo(a)pyrene (Ishinishiet aI., 1977), repeated experiments have failedto show evidence of carcinogenicity induced by arsenic compounds themselves. More recently,intratracheal administration to rodents of arsenic compounds separately and especially in combination with other agents encountered in smelter air have succeeded in producing lung tumors (reviewed in Lhonard (1984)). This lack of an animal model for arsenic-induced carcinogenesis could be indicative of differencesin the uptake and metabolism of arsenic in rodent species or itmay indicate that arsenic acts primarily as an indirect or co-carcinogen in combination with other agents (Stohrer, 1991). This mode of action would be consistent with the abilityof arsenic to inhibitD N A repair (Li and Rossman, 1989; Rossman et aI., 1977) and induce gene amplification (Barrett and Lcc, 1992). 3.2. CHROMIUM:SPECIATIONAND COMPLEXCHEMISTRY Occupational exposure to dusts or fumes containing chromium(VI) has been associated with an increased risk of lung cancer (Hayes, 1988; IARC, 1990; Langard, 1990). In a few cases, multiple primary tumors have been reported among lung cancer patients who had been workers in the chromium industry (Uyama et al., 1989). However all of the patients with multiple tumors were also heavy smokers and the actual role of chromate in the etiology of the multiple tumors is not clear. Efforts to reduce exposure to CaCrO4 during the production of chromate have resulted in significant decreases in mortality due to lung cancer in exposed populations (Korallus, 1986). Other occupational hazards due to hexavalent chromium include 'chrome' ulcers, deeply penetrating, slow healing lesions on the hands of tannery workers and in the nasal septum of chromate workers and allergic dermatitis (Langard and Norseth, 1979). These results serve to illustrate the carcinogenic potential of excess occupational exposure to chromates. In contrast to the increased risk for lung cancer among chromate workers, exposure to chromate laden soils outside of the workplace, e.g. at 'Superfund' or hazardous waste sites, has been determined to be of negligible risk for human carcinogenesis (Paustenbach et al., 1991a,b). Animals exposed to chromate salts by several routes also show an increased incidence of tumors at the site of exposure (Hayes, 1982). This lack of organotropism for chromium is the consequence of both the relatively rapid extracellular metabolism of the chromate anion, which results in the formation of inactive chromium(Ill) (De Flora et al., 1990), and the rapid cellular uptake of oxidized chromium(VI) and is indicative of the strong role chemical speciation plays in the complex toxicity of this metal. Chromium(VI) is a classical genotoxic carcinogen which tested positive in most short-term genotoxicity assays (De Flora et al., 1990). Yet the mechanisms of chromium toxicity are unclear. Chromate (Cr6+) is the biologically active species because of its rapid cellular uptake. However, once inside the cell the CP + is reduced via short-lived reactive intermediates and stable chromium(III) (Cr 3÷) complexes are produced. In the process free radical intermediates and active oxygen species may also be formed (Standeven and Wetterhahn, 1991a). It is not known which or how many of these species produce the observed genotoxic effects, or which of the measured genotoxic endpoints might be correlated with the carcinogenic process.

3.3. NICKEL: UPTAKE AND CELLULAR METABOLISM Insoluble nickel compounds are strongly carcinogenic in vitro and in vivo (Coogan et al., 1989; Costa, 1991; Sunderman, 1984a; USEPA, 1986), whereas soluble nickel compounds are weaker carcinogens. Inhalation is the primary route of human exposure to carcinogenic nickel compounds.

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E.T. SNOW

Occupational exposure to nickel fumes in nickel refining and processing is correlated with increased risk for lung cancer and an even greater risk of nasal cancer (Kaldor et al., 1986). In rodents, inhalation exposure to nickel salts produces lung cancer, while intramuscular injection of nickel particles produces tumors at the site of injection. Surprisingly, parenteral injection of soluble nickel chloride also results in lung tumors in rats and the resulting lesions mimic exposure by inhalation or intratracheal instillation (Knight et al., 1991; Sunderman et al., 1989) indicating that soluble nickel is organotropic for the lung. Nickel exposure also affects the immune system (Coogan et al., 1989). Nickel can elicit an immune response resulting in allergic contact dermatitis (Haley et al., 1987; Judde et aL, 1987; Smialowicz et al., 1987) or asthma (Malo et al., 1985; Nieboer et al., 1984). It has also been shown to be immunotoxic, with specific effects on T-cells and natural killer (NK) cells (Haley et al., 1987; Judde et al., 1987; Kasprzak et al., 1987b; Smialowicz et al., 1987). The mechanisms of nickel-induced genotoxicity are uncertain. Nickel is mutagenic in some (Chiocca et al., 1991; Christie et al., 1992; McGregor et al., 1988), but not most, mammalian mutagenesis assays and is not mutagenic in bacterial assays (Christie and Katsifis, 1990; Coogan et al., 1989). Although nickel salts by themselves are not generally mutagenic, they do act synergistically as co-mutagens in the presence of other bacterial (Dubins and Lavelle, 1986) and mammalian mutagens (Christie, 1989; Sahu et al., 1989). In mammalian cells in vitro cellular transformation by nickel is associated with phagocytic uptake of insoluble nickel species (Costa, 1991; Miura et al., 1989). Phagocytosis of nickel compounds is also associated with the release of active oxygen species by pulmonary alveolar macrophages (Zhong et al., 1990). Nickel genotoxicity is likely to be multifaceted, dependent on the mechanism of nickel uptake and related to alterations in DNA-protein interactions (Christie and Katsifis, 1990). 3.4. OTHER METALS: DIVERSITYOF ACTION 3.4.1. Beryllium Although recent studies have confirmed an association between beryllium exposure and human carcinogenesis (Steenland and Ward, 1991), the risk of increased lung cancer due to beryllium exposure is low and overshadowed by the much greater increased risk of pneumoconiosis and death due to acute beryllium disease (Steenland and Ward, 1991). The mechanisms of beryllium-induced cellular toxicity and genotoxicity are presumed to be related to previously established effects of beryllium salts on DNA synthesis in vitro (Ashby et al., 1990; Cohen et al., 1990; Luke et al., 1975; Witschi, 1970) and the inhibition of alkaline phosphatase and other phosphate-binding enzymes (Skilleter, 1984). The validity of this linkage is unknown. Beryllium, like most other metal salts, elicits a strong cellular immune response in vivo. In fact, it has recently been confirmed that chronic beryllium disease is the result of a hypersensitivity reaction in the lungs (Kriebel et al., 1988; Reeves, 1989) 3.4.2. C a d m i u m In marked contrast to arsenic, chromium and nickel, evidence for cadmium-induced carcinogenesis in man is weak and inconclusive (Sunderman, 1984b; Waalkes and Oberd6rster, 1990). Cadmium is primarily associated with cancers of the prostate and lung, in man and of the testes, lung, prostate and injection site in animals (Waalkes and Oberd6rster, 1990). The specificity for the testes and prostate may be related in part to the lack of metallothionein in these organs. It may also be the result of cadmium-induced alterations in cellular homeostasis of essential metal ions such as copper, zinc and calcium (Staessen et al., 1991). Cadmium genotoxicity appears to be related to both DNA (Coogan et al., 1992b; Waalkes and Poirier, 1984) and protein interactions (Sunderman, 1990). Inhibition of DNA repair by cadmium can enhance the mutagenicity (Rossman et al., 1992) and carcinogenicity of alkylating agents (Wade et al., 1987).


37

4. MECHANISMS OF GENOTOXICITY AND CARCINOGENESIS BY SPECIFIC METALS 4.1. ARSENIC: DNA REPAIRAND GENE EXPRESSION 4.1.1. Bioavailability and Uptake

Arsenic is a common environmental toxicant which is found in the drinking water supply in some areas of the United States (Lronard, 1991). In a number of townships in western Taiwan the concentration of arsenic in well water has been found to exceed 0.35 ppm, a level which is associated with arsenic-related blackfoot disease and an increased incidence of cancer (Chen and Wang, 1990). Occupational exposure to arsenic has been documented as the result of smelting of other metals, application of arsenical pesticides and herbicides and generation of power from coal (Aposhian, 1989; Lronard, 1991; Nordenson et al., 1978; Stohrer, 1991). Chronic arsenic exposure is of greater concern than acute exposure. Environmental exposure to arsenic is generally in the form of either arsenate (As 5÷) or arsenite (As3+). The lower valence state, arsenite, is more toxic in vivo and in vitro. However, arsenate is similar in structure to inorganic phosphate and is known to inhibit metabolic reactions in mitochondrial oxidative phosphorylation by substituting for inorganic phosphate with subsequent formation of an unstable arsenate ester (Aposhian, 1989). Biological inactivation of arsenic involves oxidation of arsenite to arsenate and subsequent alkylation to the organic arsenic derivatives methylarsonate and dimethylarsonate which are rapidly excreted (L~onard, 1991; Marafante et al., 1985; Tam et al., 1979). Biomethylation in mammals takes place in the liver by enzymatic transfer of the methyl group from S-adenosyl methionine and the major excreted metabolite is dimethylarsonate (Aposhian, 1989; Buchet et al., 1984). Arsenite detoxification might also be achieved by protein binding (Vahter and Marafanta, 1985). Arsenate can be activated in vivo by reduction to arsenite (As3+). Arsenite exerts toxic effects by reacting primarily with thiols and with sulfhydryl groups in cellular proteins (Aposhian, 1989; Aposhian and Aposhian, 1989). In particular, As 3+ can form stable interactions with dithiol groups of proteins and enzyme cofactors. An important target for As 3÷ binding is the coenzyme lipoic acid with which it interacts to form a stable 6-membered ring resulting in enzyme inactivation (Aposhian, 1989). Lipoic acid is a crucial component of the pyruvate dehydrogenase multienzyme complex and plays an essential role in the formation of acetyl coenzyme A (acetyl CoA) and gluconeogenesis (Lehninger, 1975). Arsenite also inhibits glutathione reductase and treatment of rat kidney tubules with micromolar concentrations of As 3÷ results in a 50% decrease in both acetyl CoA and glutathione (GSH) (Szinicz and Forth, 1988). Glutathione is protective against arsenite (L~onard, 1991), suggesting that the arsenic-induced decrease in GSH plays a key role in arsenite toxicity. These alterations in substrates required for cellular intermediary metabolism may play a role in arsenite-induced stress responses which will be discussed below. 4.1.2. Inhibition of DNA Repair Processes and Mutagenesis

Arsenic compounds are not mutagenic in either bacterial or mammalian cell systems (JacobsonCram and Montalbano, 1985; Rossman et al., 1980). However arsenite is a co-mutagen; that is, it enhances the genotoxic and mutagenic response to other mutagens. This effect may also be related to arsenic-induced protein inhibition. In bacterial cells arsenic has a two-fold effect: it inhibits the induction of proteins involved in the so-called 'error-prone repair' (SOS) pathway and it enhances mutagenesis induced by u.v. (Li and Rossman, 1989) and (to a lesser extent) N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) (Nunoshiba and Nishioka, 1987). The SOS repair pathway in E. coli is an inducible response to DNA damage that involves the coordinate regulation of DNA repair genes and genes required for mutagenic bypass of DNA lesions (Kenyon, 1983). Unlike many other carcinogens arsenic does not induce the SOS response (Rossman et al., 1984); in contrast, sodium arsenite actually inhibits the induction of specific SOS genes including umuDC which is required for error-prone mutagenic bypass of both spontaneous and induced DNA damage (Nunoshiba and Nishioka, 1987). As a consequence arsenite decreases u.v.-induced mutagenesis in cells lacking

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E.T. SNOW

normal u.v. repair (uvrA ") (Nunoshiba and Nishioka, 1987) and enhances u.v. mutagenesis in wild type cells due to inhibition of DNA repair (Rossman, 1981). As mentioned above, arsenite also increases the mutagenicity of methylating agents such as M N N G in bacterial cells (Nunoshiba and Nishioka, 1987). This effect could possibly be the result of a weak inhibition of O6-alkylguanine DNA alkyltransferase (AGT) by arsenic. AGT is an inducible protein responsible for the primary repair of O6-methylguanine, one of the most important mutagenic and carcinogenic lesions induced by M N N G and other alkylating agents (Saffhill et al., 1985). The active site of all known AGT proteins contains an essential cysteine which acts as an acceptor for the alkyl group from the O6-position of guanine (and, in the case of the E. coli ada protein, will accept the alkyl group from the O4-position of thymine, as well). The human, rat and bacterial AGT proteins are strongly inhibited by other metals which react with thiol groups, especially Cd 2+, Cu 2+, Zn 2+ and Hg 2+ (Bhattacharyya et al., 1988). The rat AGT protein is also weakly inhibited by arsenite, however the bacterial protein has not been tested for inhibition by this metal (Scicchitano and Pegg, 1987). In summary, arsenic-induced alterations in bacterial genotoxicity appear to be dependent on the inhibition of specific DNA repair enzymes. Arsenite is also comutagenic but not mutagenic by itself in mammalian cells. Although arsenic compounds caused both cell transformation and cytogenetic effects in Syrian hamster embryo (SHE) cells, neither arsenate nor arsenite produced mutations in either the hypoxanthine phosphoribosyltransferase (hprt) or the Na ÷, K+-ATPase genes (Lee et al., 1989). Arsenic is also nonmutagenic in Chinese hamster V79 cells (Rossman et al., 1980). However, either pre- or post-treatment incubation with arsenite enhances mutagenicity by the alkylating agent methylnitrosourea (MNU) as well as both near and far u.v. light in V79 cells (Li and Rossman, 1989, 1991). Arsenite is also comutagenic with u.v., methyl methanesulfonate (MMS) and cis-diamminedichloroplatinum (cis-Pt) at the hprt locus in Chinese hamster ovary (CHO) cells (Lee et al., 1986, 1985). In contrast to the negative results in Chinese or Syrian hamster cells, toxic concentrations of arsenite produced a low, but significant, increase in mutation frequency at the thymidine kinase (tk) locus in the mouse lymphoma L5178Y cell line (Oberly et al., 1982). High concentrations of arsenate also gave a positive response in the same system, but only after metabolic activation by rat liver $9 fraction (Oberly et al., 1982). It is probable that activation of arsenate entailed reduction to the A s 3+ species. The mouse lymphoma tk +/ mutagenesis assay is sensitive to many clastogenic mutagens that do not give a positive response in hprt mutagenesis assays (DeMarini et al., 1989). A positive response in tk assays where none is seen at the hprt locus may indicate that arsenite mutagenesis, especially at toxic doses, is the result of clastogenesis which may give rise to rearrangements or deletions rather than point mutations. Recently, Li and Rossman (1989) reported that arsenite inhibits DNA ligase II, an enzyme required in the final stages of DNA excision repair. DNA ligase activity is inhibited in vitro by as little as 10 pM arsenite (Li and Rossman, 1989). Inhibition of DNA excision repair at this rejoining step could account for both the co-mutagenic and clastogenic activities of arsenite in mammalian cell systems (Li and Rossman, 1989). It is likely that inorganic arsenic compounds also act as cocarcinogens rather than primary carcinogens in vivo. 4.1.3. Chromosomal Effects

Although dimethylarsenic acid has been reported to produce DNA strand breaks in the presence of hydrogen peroxide (Yamanaka et al., 1989), arsenite has not been shown to cause direct chemically-induced DNA damage such as DNA strand breaks. Inorganic arsenic salts do, however, induce alterations in chromosomal structure and are clastogenic in a number of systems. Arsenic compounds produce both chromosomal aberrations and sister chromatid exchanges (SCE) in mammalian cells in culture (Jacobson-Cram and Montalbano, 1985). Arsenite and, to a lesser extent, arsenate produce chromosome aberrations and SCE in SHE cells (Lee et al., 1985). Arsenite also produces a small increase in chromosomal aberrations in actively dividing CHO cells; moreover, post-treatment of cells with sodium arsenite synergistically enhances the production of chromosomal aberrations induced by ethyl methanesulfonate (Huang et al., 1986). Peripheral blood lymphocytes of workers occupationally or pharmaceutically exposed to relatively high concen-


39

trations of arsenic have also been reported to show excess chromosome aberrations and SCE induction; however, these studies on exposed human populations all have serious flaws which have previously been discussed (Jacobson-Cram and Montalbano, 1985). The chromosomal effects of arsenite are most likely mediated by effects on DNA repair enzymes such as DNA ligases (Li and Rossman, 1989).

4.1.4. Gene Amplification and Expression In addition to its comutagenic and cytogenetic effects, arsenic also induces gene amplification and alterations in gene expression. Arsenic compounds induce dose-dependent gene amplification of the essential dihydrofolate reductase (dhfr) gene in SHE cells (Lee et al., 1988) and induce amplification of the dhfr gene, but not SV40 genes, in SV40-transformed human keratinocytes (Woloson, 1990). In addition, arsenite failed to cause amplification of SV40 sequences in Chinese hamster CO60 cells (Li, 1989). Arsenite resistant trypanosomes (Leishmania species) have also been found to contain multiple amplified genes, including a 2-kb coding region of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene (Katakura and Chang, 1989). These results suggest that the mechanisms involved in the amplification of endogenous genes must differ from that of integrated but exogenous SV40 or other viral genes. It is likely that dhfr amplification is the result of arsenite-induced alterations in cellular metabolism which lead to feedback or stress induced amplification of this essential gene. This mechanism is consistent with methotrexateinduced amplification of the dhfr gene, which is the result of direct inhibition of the D H F R enzyme itself (Schimke, 1980). Amplification of exogenous, non-essential, SV40 DNA sequences has been shown to be induced by DNA damage (Lavi, 1981), but is unlikely to result from enzyme inhibition or generalized cell stress. In addition to its effects on gene amplification, arsenite has been found to induce gene expression of a number of stress response proteins. Exposure of cells to elevated temperatures or other forms of stress, including arsenite, which can cause denaturation of cellular proteins (Ananthan et al., 1986) results in the expression of a series of highly conserved heat shock proteins (hsp). Arsenite induces a number of these proteins (Deaton et al., 1990; Otsuka et al., 1990) including: ubiquitin, which is involved in protein turnover; hsp90, associated with signal transduction pathways; hsp70, a family of proteins associated with protein translocation across membranes; and heme oxygenase (hsp23), which is thought to protect against oxidant damage and is also induced by cadmium chloride, UVA light and hydrogen peroxide, substances which have the ability to reduce intraceUular glutathione levels (Keyse and Tyrrell, 1989). The inclusion of ubiquitin on this list is of interest because tad6, a yeast DNA repair gene, has been identified as a ubiquitin-conjugating enzyme (Prakash, 1989). Thus arsenic-inducible genes are related in at least one way with DNA repair in eukaryotic cells. Arsenite has also been shown to increase expression of the multidrug resistance gene (Chin et al., 1990) and c-fos (Gubits, 1988). Gene amplification and overexpression have been found to be effective mechanisms for activation of protooncogenes and are important components of tumor progression (Schwab, 1990); they may also be involved in arsenic-related carcinogenesis. In summary, it appears that carcinogenesis induced by inorganic arsenic is the result of alterations in cellular metabolism due to the inhibition of essential proteins and to gene amplification which can lead to increased expression of several known cancer-related genes. 4.2. CHROMIUM:REDOXPROCESSINGAND DNA INTERACTIONS 4.2.1. Bioavailability and Uptake Chromium is both an environmental toxicant and an essential trace metal. The glucose tolerance factor is an example of a chromium-containing protein which is required for the optimum activity of insulin (Anderson et al., 1991). Occupational exposure to excess chromium is, however, also associated with an increased risk for lung cancer (IARC, 1987). Chromium exists in a number of oxidation states, of which only Cr(VI) and Cr(III) are biologically and environmentally stable (Fishbein, 1981). Chromium(III) is present in greatest abundance in the environment; however, chromium(VI), as chromate, is the primary toxic form. Most chromium(III) compounds are not

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taken up by ceils and extracellular reduction of Cr(VI), such as in the gastrointestinal tract, tends to reduce its biological activity (De Flora et al., 1990). In contrast to Cr(III), Cr(VI), as chromate, mimics essential phosphate and sulfate anions and enters cells through general anion transport channels (De Flora and Wetterhahn, 1989). The uptake of chromate follows saturation kinetics. Once inside the cell Cr(VI) is rapidly reduced through relatively unstable Cr(V) and Cr(IV) intermediates to kinetically stable Cr(III) species (Bjerrum and Bjerrum, 1990). Intracellular chromate reduction is required for chromium compounds to exert their genotoxic effects. However, it is uncertain which intracellular forms of chromium or byproducts of chromium reduction are the ultimate carcinogenic and/or mutagenic species (Alcedo and Wetterhahn, 1990; Bianchi et al., 1983; De Flora et al., 1990; De Flora and Wetterhahn, 1989). 4.2.2. In vitro Cell Transformation Morphological transformation of cells in culture has long been used as a surrogate to measure carcinogenic potential of chemical agents (Berwald and Sachs, 1965; Borek and Sachs, 1966). Chromium compounds, like other metal carcinogens, can effectively transform human or rodent cells in culture and induce focus formation (DiPaolo and Casto, 1979; Elias et al., 1991; Fradkin et al., 1~975;Patierno et al., 1988), anchorage independence (Biedermann and Landolph, 1987, 1990; Briggs and Briggs, 1988; Elias et al., 1991; Fradkin et al., 1975; Patierno et al., 1988) and tumorigenesis (Briggs and Briggs, 1988). Somewhat surprisingly, low concentrations of chromium(III) compounds are also able to induce morphological transformation of diploid human fibroblasts, possibly due to Cr(III)-induced membrane damage (Biedermann and Landolph, 1990). Chromium(III)-induced cell transformation, unlike that induced by chromium(VI) compounds, is not associated with increased mutagenesis (Biedermann and Landolph, 1990). Not only is the valence state of chromium important, the solubility and the cationic component of the chromium salt also make a difference. For example, 'insoluble' lead chromate was better able to induce morphological transformation than soluble chromium(VI) compounds (Patierno et al., 1988). More recently, it has been shown that the transforming potency of various chromium salts corresponds to the relative intracellular chromium concentration (Elias et al., 1991, 1989) and that the insoluble salts are dissolved slowly by the culture medium prior to effective uptake (Elias et al., 1991). In addition, the lead component of 'insoluble' lead chromate acts synergistically to increase the transformation induced by chromium alone (Elias et al., 1991), suggesting that these two metals act in different ways to promote genotoxicity in vitro. Chromium(VI), but not chromium(III), also produces alterations in intercellular communication (Mikalsen, 1990) and can act as a promoter of chemically-induced cell transformation (Rivedal and Sanner, 1981). Intercellular communication and signal transduction are important components of tumorigenesis and tumor promotion. 4.2.3. D N A and C h r o m o s o m a l D a m a g e Chromate produces numerous types of genetic damage in cultured mammalian cells: chromosome aberrations (Bianchi et al., 1983; Sugiyama et al., 1991a), sister chromatid exchanges (Bianchi et al., 1983; Levis and Bianchi, 1982), DNA strand breaks (Bianchi et al., 1983; Snyder, 1988; Sugiyama et al., 1989a,c, 1986a), oxidized base damage (Aiyar et al., 1989), DNA-DNA and DNA-protein crosslinks (Borges et al., 1991; De Flora and Wetterhahn, 1989; Sugiyama et al., 1986b; Tsapakos et al., 1983; Tsapakos and Wetterhahn, 1983) and mutations (De Flora and Wetterhahn, 1989; Hartwig and Beyersmann, 1989b; Levis and Bianchi, 1982; Sugiyama et al., 1991a). As will be discussed below, the proportion of the different types of damage may vary depending on the treatment conditions and the pathway of chromate reduction. At the chromosomal level, chromium is a strong clastogen and produces both chromosome aberrations and sister chromatid exchanges. Chromatid damage, especially chromatid breaks and gaps, are induced at high frequency by soluble and weakly soluble compounds of chromium(VI). At higher Cr(VI) concentrations, chromatid exchanges are also seen (Levis and Majone, 1979). SCEs are induced by low concentrations of chromium(VI) both in vitro and in vivo, e.g. in the lymphocytes of chromate exposed workers (Stella et al., 1982). Surprisingly, SCE induction in exposed populations is not dose dependent; young workers with less than 1 year of occupational


41

exposure have been reported to have higher SCE levels in their peripheral blood lymphocytes than older workers with longer periods of exposure (Stella et al., 1982). This data suggests that there may be some mechanism of biological adaptation to chronic chromium toxicity. 4.2.4. Bacterial and Mammalian Cell Mutagenesis Chromium(VI) compounds are mutagenic in both prokaryotic and eukaryotic cells in culture (Bianchi et al., 1983; De Flora et al., 1990; Hansen and Stern, 1984a). Chromate induces base substitution mutations in E. coli (Nakamuro et al., 1978; Nestmann et al., 1979; Venitt and Levy, 1974) and, as with many mutagens, the response is strongest at toxic doses (less than 10% survival). Results of mutagenesis experiments in various Salmonella strains indicate that chromium induces more base substitution mutations than frameshift mutations (Nestmann et al., 1979; Petrilli and De Flora, 1977), more mutations at AT sequences than at GC sequences (Bennicelli et al., 1983) and that chromate-induced base substitution mutagenesis is more efficient in the presence of the plasmid pKM101, i.e. it requires SOS-dependent 'error-prone repair'--which is a function of the plasmid-encoded mucAB gene product (Nestmann et al., 1979; Petrilli and De Flora, 1977). Surprisingly, many chromium(VI) compounds are only moderately mutagenic (less than a 10-fold increase over background) in some but not all mammalian mutagenesis assays (Bianchi et al., 1983; Biedermann and Landolph, 1990; Celotti et al., 1987; Newbold et al., 1979). As with mammalian cell transformation assays, the less water soluble chromium compounds may be more potent in producing mammalian cell mutagenesis (Patierno and Landolph, 1989). For example, the poorly soluble calcium chromate (CaCrO4) induced a high frequency of 6TG R mutants in human fibroblast cells (HFC) (Biedermann and Landolph, 1990) and induced both 6TG R and Oua R mutants in CHO cells (Patierno and Landolph, 1989). Na +, K+-ATPase is an essential gene and Oua R mutations occur only by base substitution (Muriel et al., 1987); thus, this chromium compound, at least, can induce base substitution mutations in mammalian cells. Both potassium dichromate (K2Cr207) and potassium chromate (K2CrO4) are strong mutagens in the mouse lymphoma L5178Y/tk +/ assay (Oberly et al., 1982). This assay is especially sensitive to mutagenesis by clastogens (DeMarini et al., 1989) and the results may indicate that chromate induces a high frequency of deletions or rearrangements. However, chromate damage is not radiomimetic and it does not induce a high frequency of mutagenesis at all gene targets which are sensitive to clastogens. We have recently characterized two independently-derived transgenic V79 cell lines with integrated bacterial gpt genes and used them to assay chromate-induced mutagenesis at the gpt locus (6TGR). These cell lines are especially sensitive to mutagenesis by X-rays, bleomycin and other elastogenic agents (Klein and Rossman, 1990; Klein and Snow, 1991a,b). We find that chromate, unlike X-rays or even u.v. light, is only a moderate mutagen in these cells, as it is in V79 cells at the hprt locus (Celotti et al., 1987; Elias et al., 1986; Sugiyama et al., 1991a). In our hands, chromate induces no more than a 5- to 10-fold increase in mutation frequency above background (Klein et al., 1992). It should also be noted that chromate induces persistent toxicity and at the higher doses the plating efficiency of the treated cells is reduced even one week after treatment. This residual effect may contribute to a selective loss of mutant cells and to the frequently observed decreased mutant yield at higher doses. This also implies that there is continued damage produced or persistent chromium adducts which exist for some time after initial exposure. Chromate, like other metal salts which induce persistent toxicity, exhibits a narrow effective dose range. 4.2.5. Mechanisms of Chromium Mutagenesis The mutagenic DNA lesions induced by chromate have not yet been established. Both chromium(III) and chromium(VI) have been surprisingly refractory in producing mutagenic DNA damage in cell free systems (Bianehi et al., 1983; De Flora and Wetterhahn, 1989). It is possible that the reduced chromium species themselves are the biologically active promutagenic species or ligands. It has been postulated, for example, that chromium(V) intermediates facilitate the production of oxidative damage within the cell and that chromium(V)--GSH conjugates may be involved in DNA-protein crosslinking (De Flora and Wetterhahn, 1989). It is known that chromium can produce both base substitution and frameshift mutations in Salmonella assays

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(Bianchi et al., 1983; L~onard and Lauwerys, 1980; Levis and Bianchi, 1982) and can produce base substitution mutations in E. coli (Bianchi et al., 1983; L~onard and Lauwerys, 1980). It is not known what specific types of base substitutions or frameshifts are produced or what species of chromium are responsible. The types of mutations induced by chromium in mammalian cells are almost completely unknown. The production of oxidative species is one mechanism by which chromium may produce mutagenic DNA damage that contributes to chromate carcinogenesis (De Flora and Wetterhahn, 1989). Oxidative DNA damage has been implicated in chromate mutagenesis in several ways: by increased chromate mutagenesis in AT-specific Salmonella reversion strains (TAI02) compared with that in GC-specific strains (TA100) (Bennicelli et al., 1983; De Flora and Wetterhahn, 1989); by the formation of 8-oxo-2'-deoxyguanosine (8-OHdG), and, presumably, other oxidative DNA damage, in chromium-treated DNA in vitro (Aiyar et al., 1989) and in vivo (Liebross and Wetterhahn, 1990); and by the formation of redox active Cr(V) species in vitro (Aiyar et al., 1989; Alcedo and Wetterhahn, 1990; De Flora and Wetterhahn, 1989; Sugiyama et al., 1989a,c). Oxidized base lesions are known to be mutagenic in bacterial systems (Basu et al., 1989; Floyd, 1990; Hayes et al., 1988; Morita et al., 1991; Shirname-More et al., 1987) and to be genotoxic in mammalian cells (Kaufman, 1989; Shirname-More et al., 1987). Oxidized pyrimidines (e.g. thymidine and cytosine glycols) may block DNA replication and have been shown to produce T ~ C (Basu et al., 1989) and C--*T (Hayes et al., 1988) base substitutions. 8-OHdG produces mutations (albeit at low efficiency) (Floyd, 1990; Morita et al., 1991). Exogenous 5-hydroxymethyl2'-deoxyuracil (HMdU) can be mutagenic (Shirname-More et al., 1987) and can also produce sister chromatid exchanges in mammalian cells (Kaufman, 1989). Other types of DNA damage induced by chromium may also be mutagenic. DNA strand breaks may lead to the production of deletion mutations and gene rearrangements (Grosovsky et al., 1988; Liber et al., 1989). DNA-protein crosslinks (DPC) may also promote DNA recombination and/or deletion mutagenesis by acting as blocks to DNA replication. Repair of DPCs is significantly slower than repair of DNA strand breaks or interstrand cross-links (Sugiyama et al., 1986a,b). Chromiuminduced DNA damage does not appear to be repaired by bulky lesion excision repair (Snyder et al., 1989). 4.2.6. The Role o f C h r o m i u m ( I I I ) Although chromium(III) compounds are not significantly toxic to intact cells, Cr(III) is more effective than Cr(VI) in genotoxicity assays using cell-free systems. In fact, chromium(II), which becomes Cr(III) in aqueous solution, is more effective than chromium(VI) in decreasing the fidelity of DNA replication in vitro (Sirover and Loeb, 1976a,b). Chromium(Ill) has a 2-fold effect on DNA replication in vitro. At very low concentrations, Cr(III) species bind to the DNA phosphates and increase non-specific DNA (Snow, 1991; Snow and Xu, 1989, 1991) and RNA (Niyogi and Feldman, 1981; Niyogi et al., 1981) polymerase activity. DNA polymerases bind more tightly to chromium(III) modified templates and incorporate more nucleotides per binding event (processivity is increased) (Snow and Xu, 1991). This more processive mode of polymerase activity is also more error prone (Snow and Xu, 1991), allows increased bypass of oxidative DNA damage (E. T. Snow and L.-S. Xu, unpublished results) and may promote damage-specific misincorporation. DNA-bound chromium(III) could contribute to the greater than additive mutagenesis induced by chromium(VI) in the presence of a second mutagen (LaVelle, 1986; LaVelle and Witmer, 1984). In addition to its effects on DNA polymerase processivity, chromium(III) bound to the DNA can also contribute to the production of DNA strand breaks (Ozawa and Hanaki, 1990; Sugden et al., 1990). Chromium(III)-DNA-protein complexes have been shown to interfere with the activity of DNA restriction enzymes (Borges and Wetterhahn, 1991; Chen et al., 1991) and, although they have been called 'kinetically inert', chromium(III) complexes can act as catalysts to coordinate the self polymerization of olefins (Thomas and Theopold, 1988). Likewise, chromatin-associated chromium(III) complexes may play an active role in the genotoxicity of chromium in vivo.


43

4.2.7. Metabolic Modulation o f Chromium Genotoxicity Although it is known that chromium is genotoxic, it is still not known exactly why or how. The difficulties involved in understanding the mechanisms of chromium-induced genotoxicity are threefold: (i) Cr(VI) compounds but not Cr(III) compounds are readily transported across cell membranes; therefore, only Cr(VI) produces genotoxic effects in experimental whole cell systems, (ii) Cr(VI), once inside the cell, is readily reduced to Cr(III) via reactive Cr(V) and Cr(IV) species and ligands and (iii) Cr(III) is more active than Cr(VI) in DNA binding assays and in assays measuring the fidelity of DNA replication in cell-free systems. Thus, it is not known which aspects of chromium toxicity are mediated by reactive Cr(V) or Cr(IV) species (Alcedo and Wetterhahn, 1990; De Flora and Wetterhahn, 1989), which are due to stable Cr(III) interactions (Ozawa and Hanaki, 1990; Snow, 1991; Snow and Xu, 1989, 1991), or which are the result of oxidative damage produced by free radicals or reactive oxygen species generated during chromate reduction (Aiyar et al., 1991; Shi and Dalai, 1990; Standeven and Wetterhahn, 1991a), Redox interactions and DNA crosslinks resulting from chromium reduction (including oxidative base damage) are generally considered to be the most likely causes of chromate genotoxicity, however this remains to be proven. The intracellular reduction of chromate can occur by several different pathways, each of which may produce different toxicological endpoints (Alcedo and Wetterhahn, 1990; Conner and Wetterhahn, 1983; Cupo and Wetterhahn, 1985). The reduction of chromium(VI) can be accomplished by various microsomal enzymes (including cytochrome P450 (Mikalscn et al., 1989, 1991b; Mikalsen, 1990), glutathione reductase (Conner and Wetterhahn, 1983) and components of the mitochondrial electron transport system (Arillo and Melodia, 1988; Arillo et al., 1987)), glutathione (Standeven and Wetterhahn, 1989) and other small molecular weight non-protein thiols (Connett and Wetterhahn, 1983, 1985; De Flora and Wetterhahn, 1989), ascorbic acid (Standeven and Wetterhahn, 1991a,b; Sugiyama et al., 1991b; Suzuki, 1990; Suzuki and Fukuda, 1990), riboflavin (vitamin B:) (Sugiyama et al., 1989a,c) and others. Intracellular reduction of chromium results in the transient formation of chromium(V) species and the eventual accumulation of reduced chromium(III) within the mitochondria (Langard, 1979) and the nucleus (Langard, 1979; Sehlmeyer et al., 1990), where presumably most of the chromium is bound to the DNA and nuclear proteins. Conner and Wetterhahn (1983) were the first to note that the intracellular metabolism of chromate is under kinetic rather than thermodynamic control. This means that the most plentiful and kinetically active electron donors will be of primary importance in the reduction of chromate. Enzymatic reduction of chromium(VI) by microsomal enzymes or the mitochondrial electron transport system is slow and therefore of less overall importance than reduction by various small compounds such as glutathione and ascorbic acid which can be present in millimolar concentrations. However, microsomal or mitochondrial reduction of chromate can result in the reduction of key cellular metabolites such as ATP and NAD(P)H (Standeven and Wetterhahn, 1989) and the inactivation of crucial enzymes, such as DT-diaphorase (Arillo et al., 1987), glutathione reductase (Sugiyama et al., 1989b) and g-ketoglutarate (Ryberg and Alexander, 1990). Mitochondrial reduction of chromate and the subsequent accumulation of Cr(III) can also result in the loss of mitochondrial DNA and proteins (Egilsson et al., 1979). Cellular toxicity and mitochondrial damage resulting in cell death due to loss of cellular redox capacity can lead to the preferential loss of damaged cells. Since dead cells cannot give rise to mutant or transformed progeny, this selective killing can be thought of as antimutagenic or anticarcinogenic. Evidence for the importance of excess cellular toxicity can be seen in the observed reduction of survival and mutagenesis in cell populations treated with high doses of chromium in vitro (Klein et al., 1992). Glutathione, the tripeptide y-glutamylcysteineglycine(GSH), is an important cellular reductant present in millimolar concentrations which can bind to and promote metabolism and excretion of many toxicants. GSH can rapidly and efficiently bind chromate ions to form a Cr(VI)GSH thioester intermediate (Brauer and Wetterhahn, 1991). The chromium is then reduced in a two step process, from chromium(VI) to chromium(V); and, in the presence of excess GSH, from Cr(V) to Cr(III) (Conner and Wetterhahn, 1985; Shi and Dalai, 1988; Wetterhahn et al., 1984). Both the Cr(V)- and Cr(III)-GS complexes can apparently undergo further ligand exchange

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and eventually form Cr(III)-DNA or Cr(III)-protein complexes. Increased intracellular levels of GSH produced by N-acetylcysteine increase chromium-induced DNA-protein crosslinking (Cupo and Wetterhahn, 1985) and protein-associated single-strand breaks; but there is no evidence, as yet, for the involvement of these lesions in chromate-induced mutagenesis in mammalian cells. Isopentanol increases the levels of both GSH and cytochrome P450 in chick embryo cells and also increases chromium-mediated DNA damage (Cupo and Wetterhahn, 1985). In contrast, reducing glutathione levels by pretreatment of cells with buthionine sulfoximine results in decreased chromate-induced DNA damage in chick embryo cells (Cupo and Wetterhahn, 1985). Other cellular thiols in addition to GSH can bind and reduce chromium(VI), the rate of the reaction being inversely related to the pKa of the thiol group (Borges et al., 1991). These various chromium-thiol complexes can interact with DNA and may play a role in chromium genotoxicity (Borges et al., 1991). Other cellular reactants can also interact with chromium(VI) to produce genotoxic damage. Chromium(VI) in the presence of hydrogen peroxide can form tetraperoxochromium(V) and lead to the formation of hydroxyl radicals ('OH) which may promote DNA strand breaks and the production of oxidized base damage (Shi and Dalai, 1990; Standeven and Wetterhahn, 1991a). Vitamins can be used to treat cells in vitro and in vivo to alter cellular redox status. Such treatments can change the cellular response to chromium. For example, pretreatment of V79 cells with the anti-oxidant ~-tocopherol (vitamin E) reduces both chromate toxicity (Sugiyama et al., 1989b, 1991a) and mutagenesis at the hprt locus (Sugiyama et al., 1991a). This suggests that oxidative damage is partly responsible for chromate mutagenesis and that reduction in oxidative base damage may parallel reduction in toxicity and mutagenicity mediated by pretreatment with antioxidants. Vitamin B2 (riboflavin) increases chromium genotoxicity in the form of DNA single strand breaks (Sugiyama et al., 1989a,c) by increasing the formation (or lifetime) of chromium(V) species. This also may result in the production of increased levels of oxidized base damage and increased mutagenesis. Perhaps the most important reductant of chromium is ascorbic acid. Ascorbate is present at high (mmol) concentrations within cells. It is also present in extracellular fluids, most notably in the lung. Extracellular ascorbic acid serves to detoxify chromium(VI) by decreasing its cellular uptake (Samitz, 1970; Suzuki and Fukuda, 1990). On the other hand, the reduction of chromate by ascorbic acid inside the cell and the resultant formation of intracellular active oxygen species, increases chromium toxicity (Standeven and Wetterhahn, 1991b). Ascorbic acid and glutathione act synergistically to increase the rate of chromate metabolism (Suzuki, 1990). Sugiyama et al. (1991b) have shown that incubation of Chinese hamster V79 cells with ascorbic acid can lead to increased intracellular accumulation of vitamin C, which makes the cells more sensitive to subsequent treatment with chromate. Increased intracellular ascorbate also results in increased chromate-induced DNA-protein crosslinks and decreased production of alkali labile sites. In the presence of increased ascorbate there is less formation of chromium(V) and more Cr(III) (Sugiyama et al., 1991b). The contribution of intracellular reduction of chromate by ascorbic acid to in vivo mutagenesis or carcinogenesis is unknown. Each of the major pathways of chromate reduction results in a different spectrum of cellular and/or genotoxic damage. Enzymatic reduction of chromium, though slow relative to reduction by cellular thiols or diols, tends to result in cellular toxicity via inactivation of key enzymes, such as glutathione reductase, or by decreasing one or more cellular metabolites, such as GSH or NAD(P)H. Chromate reduction by non-protein thiols such as glutathione, however, results in the transient formation of Cr(V)-thioesters and thiyl radicals (De Flora and Wetterhahn, 1989). Under some conditions these intermediates may lead to the production of additional free radicals which may result in the production of DNA strand breaks or alkali labile sites. A major end product of thiol-mediated chromate reduction appears to be the formation of DNA interstrand crosslinks and DPCs. Although the role of these lesions in mutagenesis or carcinogenesis is unknown, the persistent nature of DPCs would suggest that they may be toxic. Reduction of chromate by ascorbic acid also seems to result in an excess production of DNA-protein crosslinks, but fewer alkali labile sites (Sugiyama et al., 1991b). Reduction in the presence of hydrogen peroxide and the formation of a tetraperoxychromium(V) intermediate gives rise to excess alkali labile sites and oxidative base


45

damage; however, this does not appear to be an important pathway for chromium reduction in vivo (Standeven and Wetterhahn, 1991a). We still have much to learn regarding mechanisms of intraceUular chromium metabolism and chromium-induced genotoxicity. Chromate is a complex environmental toxin which has diverse effects on cellular metabolism and genotoxicity. In similarity to numerous organic carcinogens which require cellular 'activation', the chromium is first activated then, in turn, it reacts with numerous cellular constituents. Like many xenobiotics that are metabolized by inducible cytochrome P450 enzymes, chromate can alter the expression of the P450 genes and thus interfere with cellular oxidative metabolism (Hamilton and Wetterhahn, 1989; Mikalsen et al., 1991a). The genotoxic damage induced by chromium is complex and not completely defined. Understanding the mechanisms of chromium genotoxicity which are important in carcinogenicity will require continued and coordinated research in inorganic chemistry, biochemistry and cell biology.

4.3. NICKEL: PROTEIN INTERACTIONSAND CELLULAR RESPONSES The mechanisms of nickel toxicity and carcinogenesis have been discussed in several excellent, recent reviews (Christie and Katsifis, 1990; Coogan et al., 1989; Costa, 1991). This section will focus primarily on newer findings and will attempt to unify the research in several different areas into a single theory of nickel carcinogenesis.

4.3.1. Uptake and Bioavailability Human populations are exposed to many forms of nickel, in the air, in food and drinking water and in numerous consumer products. Nickel allergy is the most common adverse effect arising from nickel exposure (Fisher, 1985; Peltonen, 1979; Schubert et al., 1987). Inhalation exposure in the work environment is most likely to cause significant adverse effects, including an elevated risk of lung cancer and cancer of the nasal passages (Christie and Katsifis, 1990; Coogan et al., 1989). In addition, iatrogenic exposure can occur due to surgical or dental implants and hemodialysis (Grandjean, 1984; Sunderman, 1983). Kidneys and lungs are the primary organs in which nickel is accumulated and they are the main sites of nickel damage in vivo. In general, however, the toxicity of nickel is dependent on the route of exposure and the solubility of the nickel compound. The chemical nature of a nickel compound is one of the key determinants of its toxicity (Costa, 1991). Insoluble or weakly soluble crystalline nickel compounds are more toxic than soluble nickel salts. Exposures to water soluble salts of nickel are characterized by rapid excretion, compared with exposures to sparingly soluble nickel compounds, such as nickel oxides and sulfides, which exhibit slow removal. Some of the insoluble compounds have a biological half-life of up to 3 years (Cohen et al., 1990). The availability of Ni :+ to tissues and the biological consequences of intracellular accumulation, are much greater for insoluble crystalline compounds which are phagocytized compared to soluble or amorphous compounds which are not readily phagocytized. The genotoxicity of nickel has at least two components, the effects due to the Ni 2+ ion, which can interfere with the utilization of essential divalent metals such as Mg2* and those related to the particulate nature and phagocytic uptake of some insoluble nickel compounds. A third, less defined, component of nickel toxicity involves nickel interactions with cellular macromolecules. These interactions are crucial to an understanding of the nature of cellular immune responses to nickel, the consequences of phagocytic processes, and, possibly, the ability of nickel complexes to undergo redox cycling between Ni 2+ and Ni 3+. Poorly soluble nickel compounds, such as Ni3S2, NiSO4 and Ni(CO)4, are highly carcinogenic in rats and mice. Inhalation exposure to Ni3S2 in rats (Ottolenghi et al., 1974; Sunderman, 1984a) causes hyperplasia, metaplasia, adenomas and adenocarcinomas equally in both males and females, in both bronchiolar and alveolar regions of the lung. Nickel salts are not tumorigenic following ingestion; however, intra-muscular injection of particulate nickel compounds can induce tumors at the site of injection. Tumorigenesis correlates strongly with the production of erythropoiesis by particulate nickel compounds and with phagocytic index and nickel mass fraction of the compound (Costa and Heck, 1984; Sunderman et al., 1987). J'PT 53/1--D

46

E.T. SNOW

The biological activity of inorganic nickel is ultimately due to the intracellular accumulation of soluble nickel. Absorbed nickel is systemically distributed then eliminated and the toxicological impact of nickel compounds is linked to the toxic action of the Ni 2+ ion (Costa and Mollenhauer, 1980; Hansen and Stern, 1984b). Soluble nickel compounds freely enter cells, but are just as readily excreted. Phagocytosis of insoluble nickel compounds, such as Ni 3$2 (Costa et al., 1981, 1982, 1980; Evans et al., 1982), allows the metal to interact productively with cellular macromolecules, prevents its excretion and possibly prolongs the opportunity for continued production of potential genotoxic damage (Christie and Katsifis, 1990; Costa, 1991). Costa (1991) has shown that selective phagocytosis of particulate crystalline NiS and Ni3S2 is due to negative surface charge and is dependent on both temperature and calcium ions. Ionic nickel appears to inhibit the phagocytic process (Heck and Costa, 1983; Sunderman et al., 1989). Once taken up by the cells the Ni(II) ions are released from phagocytic vacuoles on the periphery of the nucleus where they can preferentially interact with heterochromatic regions of the chromatin, producing DNA-protein crosslinks and, to a lesser extent, DNA strand-breaks. It has been recently shown that once inside the nucleus of rat lymphocytes, the nickel component of nickel sulfides, regardless of their method of uptake, is no longer associated with sulfur; it is now complexed with phosphorus, probably in association with DNA and (phosphorylated) chromatin proteins (Hildebrand et al., 1991). Carcinogenesis may result from DNA damage, from nickel interactions with enzymes involved in DNA replication or repair, or may be the result of alterations in (regulatory) DNA-protein interactions (see below). Nickel, especially carcinogenic Ni 3$2, also elicits a strong oxidative burst response in pulmonary macrophages (Zhong et al., 1990) and promotes infiltration of macrophages at other sites of nickel deposition; this oxidative response clearly plays a role in the mechanism of nickel carcinogenesis. Nickel-induced alterations in cellular immune processes (Coogan et al., 1989) are likely to play an additional, less well defined, role in the carcinogenic process (for example see Schiffer et al., 1991; Smialowicz et al., 1987; Kasprzak et al., 1987b). 4.3.2. Cellular Transformation and Mutagenesis Both soluble and particulate forms of nickel promote morphological cell transformation in vitro (DiPaolo and Casto, 1979; Hansen and Stern, 1983). Morphological transformation of Syrian hamster embryo cells by nickel compounds is dependent on the uptake and long term sequestering of these compounds in phagocytic vacuoles and the subsequent slow release of the active Ni(II) ions into the periphery of the nucleus (Cohen et al., 1990; Costa, 1983). C3H/10T1/2 mouse embryo fibroblasts have also been used recently in a cell transformation assay with various soluble and insoluble nickel compounds (Miura et al., 1989). Soluble nickel salts were cytotoxic, but did not induce morphological transformation of these cells. Insoluble nickel compounds, in contrast, were strongly positive and produced a dose-dependent increase in frequency of transformation. These same nickel compounds did not induce base pair substitition at the Oua R locus in 10T1/2 cells when assayed under the conditions that induced cell transformation (Miura et al., 1989). Of the seven nickel-induced foci that were isolated; four cell lines exhibited anchorage independent growth and one cell line formed tumors in nude mice (Miura et al., 1989). Human kidney epithelial cells have also been immortalized and transformed to anchorage independence, but not tumorigenesis, by nickel sulfate (Haugen et al., 1989). Transfer of the v H A - r a s oncogene into the nickel-transformed cells converted the immortal cell lines to tumorigenesis (Haugen et al., 1989). As described below, one of these cell lines has since been found to contain a nickel-induced point mutation in the p53 tumor supressor gene, which was also converted to homozygosity (M~ehle et al., 1992). Klein et al. (1991 a) have also shown nickel-induced deletion or inactivation of an X-linked senescence or tumor supressor gene, see below. These results all suggest that nickel can act at an early step in the carcinogenic process. However, in contrast to their carcinogenic and transforming potential, nickel compounds are only occasionally mutagenic in vitro (Christie and Katsifis, 1990; Coogan et al., 1989; L6onard and Jacquets, 1984). Nickel alone is usually toxic but not mutagenic in prokaryotic assays; however a number of studies indicate that nickel compounds may be positive in certain mammalian assays (Amacher and Paillet, 1980; Miyaki et al., 1979; Morita et al., 1991; Nishimura and Umeda, 1979; Swierenga and McLean, 1984). Recently it has been shown that insoluble nickel compounds such


47

as Ni3 $2 and crystalline NiS may be strongly mutagenic in several different forward mutagenesis assays. Swierenga and McLean (1984), for example, have shown that insoluble and soluble nickel compounds can induce hprt mutations in rat epithelial cells in culture. It is especially interesting to note that soluble NiCI2 and dissolved Ni 3$2 induced positive responses only at toxic doses while crystalline Ni3S2 gave a strong response at nontoxic doses (Swierenga and McLean, 1984). In addition, these investigators noted effects on cytokeratin structure at mutagenic doses (sec below). Insoluble nickel compounds are also mutagenic in some Chinese hamster cells (Christie et al., 1992). The G12 assay developed by Klein and Rossman (1990) measures mutagenesis in a bacterial gene (gpt) that is stably integrated into an hprt" Chinese hamster V79 cell line. This cell line is especially sensitive to mutagenesis by oxidative clastogens such as X-rays and bleomycin (Klein and Rossman, 1990; Klein and Snow, 1991a,b) and it is possible that nickel mutagenesis occurs by similar mechanisms. Crystalline nickel sulfide is much more mutagenic than nickel subsulfid¢ in this assay (Christie et al., 1992). The mechanisms of nickel-induced mutagenesis in these systems are unknown; however, Biggart et al. (1987), Biggart and Murphy (1988) and Chiocca et al. (1991) have recently characterized a nickel-induced 70 base-pair duplication which reverts a temperature-sensitive viral mRNA splice-site mutation. It has been suggested that nickel, because of its ability to induce clastogenesis, may preferentially induce DNA deletions or rearrangements (Christie and Katsifis, 1990). However, a nickel-induced point mutation (a T ~ C transition at codon 238) in the p53 tumor suppressor gene paired with a chromosome 17p deletion (making the mutated sequence homozygous) has also been reported in a human kidney epithelial cell line immortalized by treatment with NiSO4 (Ma~hle et al., 1992). Another aspect of mammalian cell mutagenesis by nickel and other metals which tend to persist within the cell for long periods after initial treatment is delayed toxicity. Most organic genotoxins exhibit short biological or chemical half lives and the damaging species is rapidly inactivated or excreted from cells after treatment. The cells can then recover from the insult and the resultant DNA damage and mutagenesis occurs either during or shortly after the treatment period. However, nickel, chromium and other metal toxicants may persist within the cell and continue to exert genotoxic effects for long periods after treatment. Thus nickel, like chromium, exhibits both delayed toxicity and a narrow effective dose range (see for example, Morita et al., 1991; Miura et al., 1989). Although nickel compounds are weak mutagens in many mammalian and bacterial assays, nickel in combination with a second agent often gives a cooperative or synergistic response (Christie, 1989; Christie and Katsifis, 1990; Hartwig and Beyersmann, 1989b). Synergistic enhancement of cell transformation by nickel compounds has been reported numerous times (reviewed in Christie and Katsifis, 1990). Nickel synergism has also been seen for bacterial (Dubins and Lavelle, 1986; Ogawa et al., 1987) and mammalian mutagenesis assays and SCE induction (Hartwig and Beyersmann, 1989b). As discussed by Christie and Katsifis (1990), these synergistic effects may be due to nickel-induced alterations in replication or processing of DNA lesions. Possibly because of its ability to substitute for essential magnesium ions in DNA polymerases, nickel salts inhibit DNA replication and also decrease replication fidelity in vitro (Christie et al., 1991; Miyaki et al., 1977; Sirover and Loeb, 1976a, 1977). The kinetics of protein-binding and nickel-induced inhibition of DNA replication by human DNA polymerase ~ suggest that there are two types of binding sites for nickel on the polymerase and that nickel bound to the tight binding site(s) acts as a competitive inhibitor of Mg-dependent polymerase activity (Christie et al., 1991)*. However, nickel is an inefficient activator of polymerase activity and decreases the fidelity of DNA replication in vitro regardless of the presence or absence of the physiological metal activator, Mg 2+ (Christie et al., 1991; Sirover and Loeb, 1976b, 1977). Recent evidence suggests that nickel salts can inhibit the repair of u.v.-induced DNA damage in both human (HeLa) cells (Snyder et al., 1989) and V79 Chinese hamster cells (Hartwig and Beyersmann, 1989b). This inhibition of repair appears to be limited to bulky-lesion ('long patch') DNA repair (Hartwig and Beyersmann, 1989b) and is also consistent with an effect on DNA polymerase activity. Significantly, treatment of SHE cells with 10 #M insoluble NiS for 24 hr *Y. E. Chin, E. T. Snow, M. Cohen and N. T. Christie. Kinetic studies of Ni2÷ effects on DNA replication by DNA polymerase ~t, in preparation.

48

E.T. SNOW

produced a greater amount of DNA-repair replication than a 3 hr treatment with 100 #M soluble NiCI2 (Robison et al., 1984). 4.3.3. Chromosomal and D N A Damage Nickel also produces DNA strand breaks and inhibits the repair of other types of DNA damage. Treatment of CHO cells in culture with soluble NiC12 resulted in few strand breaks; however, similar treatment of isolated nucleoids (chromatin) induced significant strand breaks, indicating that Ni 2÷ is capable of producing DNA damage in vitro (Robison et al., 1984). Nickel(II) has been shown to bind to DNA and protein in vitro and can promote the formation of stable DNA-protein crosslinks (Lee et al., 1982). Interaction of nickel with DNA, especially with deoxyguanosine, may promote depurination (Datta et al., 1991; Kasprzak and Hernandez, 1989; Schaaper et al., 1987) which will result in alkali-labile sites in the DNA. As will be discussed below, nickel interactions with certain amino acids or peptides in the presence of ascorbic acid or H202 can result in the formation of active oxygen species which can also lead to the production of DNA damage (Inoue and Kawanishi, 1989; Kasprzak, 1991). Carcinogenic nickel compounds produce several types of DNA damage in vivo and in vitro. For example, Ciccarelli et al. (1981) and Ciccarelli and Wetterhahn (1982) observed DNA strand breaks and persistent DNA-protein crosslinks in the nuclei of kidney and lung cells and D N A - D N A crosslinks in the kidney cells of rats injected i.p. with nickel carbonate. Kidney is the target organ for nickel-induced carcinogenesis under these conditions. Patierno et al. (1987) also found increased DNA-protein interactions in NiC12-treated CHO cells in culture, especially in the magnesium-insoluble heterochromatin fraction. As with most nickel-induced genotoxicity, the formation of DNA-protein crosslinks and DNA strand breaks by nickel in CHO cells could be prevented by increasing extracellular magnesium concentrations (Conway et al., 1987). Nickel compounds also produce chromosomal aberrations, especially gaps, breaks and exchanges (Nishimura and Umeda, 1979; Sharma and Talukder, 1987) and SCEs can be seen at doses below those which cause chromosomal aberrations (Hartwig and Beyersmann, 1989b). Although the DNA-protein crosslinks are very labile and are presumed to be mediated by the nickel ion itself (Patierno and Costa, 1987), the exact mechanism of formation of other nickel-induced DNA lesions is unknown. 4.3.4. The Role o f Oxidative Processes One of the keys to the mechanism of nickel-mediated carcinogenesis is the enhancement of cellular redox processing by nickel. I refer the reader to two excellent reviews on the role of oxidative processes in metal carcinogenesis by Klein et al. (1991b) and Kasprzak (1991) and will briefly summarize here a few aspects of nickel-induced redox processing. Nickel elicits the production of reactive oxygen species (ROS) by at least one, possibly two mechanisms--as the result of the phagocytosis of particulate nickel compounds and through the interaction of nickel ions with protein ligands which have been shown in vitro to promote the activation of the Ni2+/Ni 3+ redox couple. Phagocytic cells such as polymorphonuclear leukocytes (PMNs) and macrophages produce large amounts of ROS (especially H:O: and superoxide anion radicals ('O~-)) in response to activation by bacteria, particulates, or tumor promoters. These ROS, in turn, can be reduced to hydroxyl radicals ('OH) by transition metals such as iron and copper through the Fenton and Haber-Weiss reactions (Klein et al., 1991b). Hydrogen peroxide can also be converted to hypochlorite (HOC1/OCr) by myeloperoxidase (Klein et al., 1991b). Sulfides of both nickel and cadmium have been reported to induce H202 production in human PMNs in vitro (Zhong et al., 1990). The magnitude of the metal-induced response was comparable to that due to the tumor promoter TPA and apparently occured via a different mechanism, since the response was significantly decreased when the cells were treated simultaneously with both TPA and nickel (Zhong et al., 1990). Lipid peroxidation has also been reported to occur in lung tissue due to activation of alveolar macrophages after parenteral injection of NiCI: in rats (Sunderman et al., 1989). At a molecular level, coincubation of calf thymus DNA with Ni3 S: or NiC12 in the presence of H202 or ascorbate can lead to the formation of oxidative base damage (8-OHdG) and depurination


49

of the DNA (Kasprzak and Hernandez, 1989). Nackerdien et al. (1991) have also recently reported the formation of multiple types of oxidative DNA base damage in isolated human chromatin treated with Ni(II) in the presence of H202. When DNA is treated with relatively high concentrations of nickel, the metal ion is bound preferentially to guanine rather than pyrimidines and the DNA phosphates are part of the ligand complex (Datta et al., 1991). This contrasts with the observed preferential formation of alkali labile sites at pyrimidines rather than purines (Kawanishi et al., 1989); however, the sites of tightest binding would not necessarily correspond to the sites of oxidative damage. Oxidative damage to DNA by nickel in the presence of proteins may be the result of the formation of redox-active nickel-protein ligand complexes. Inoue and Kawanishi (1989) have recently reported that when nickel(II) is complexed with peptides such as Gly-Gly-His, it can be activated to undergo redox cycling and decompose or dismutate H202 into the very reactive ringlet oxygen (IO') and hydroxyl radicals. Thus, insoluble nickel compounds can elicit the formation of H202 by phagocytic cells and then when complexed to the appropriate ligands it can activate the peroxide into even more reactive ROS. Since the phagocytized nickel accumulates in the vicinity of the nucleus (Costa, 1991) and can form complexes with chromatin-associated proteins (Patierno et al., 1987; Wang and Costa, 1989), it could presumably also undergo redox cycling in the vicinity of the DNA. The production of oxidative DNA damage in the target tissues (Kasprzak, 1991; Sunderman et al., 1989) strongly suggests that the production of oxidative damage is an important component of nickel carcinogenesis. 4.3.5. Interactions with Heterochromatin and Magnesium Another aspect of nickel toxicity is its ability to interfere with or substitute for protein or DNA binding by essential metal cations, such as magnesium. Many of the toxic and carcinogenic effects of nickel can be antagonized by magnesium ions and much of the known toxicity of nickel may be rationalized by its ability to interfere with the activities of the physiological divalent cations Mg, Zn, Ca and Mn (Costa and Heck, 1984; Sunderman, 1978; Waalkes et al,, 1985). Interactions with various other cations may potentiate or alleviate various symptoms of nickel toxicity (Kasprzak, 1990). One consequence of this interaction is that chromosomal damage induced by nickel preferentially occurs within heterochromatic regions of the chromatin of rodent cells in culture (reviewed by Costa, 1991). This specificity is most notable upon nickel uptake by phagocytosis (Sen and Costa, 1986) and can be eliminated by the presence of excess magnesium (Conway et al., 1987). For example, crystalline NiS (but not chromate) produces an excess of SCE in the heterochromatic regions of CHO chromosomes (Sen et al., 1987). Nickel sulfide has also been shown to induce a large number of chromatid exchanges and dicentrics and a pronounced decondensation of the heterochromatic X chromosome in CHO cells (Sen and Costa, 1986). Soluble nickel chloride (NiCl2) produced a much lower incidence of dicentrics and had less effect on the decondensation of the X, unless the NiC12 was complexed with albumin and encapsulated into liposomes, which allows it to be taken up by the cells via phagocytosis. These results suggest that specific interactions of nickel ions with heterochromatin may depend upon the mechanism of exposure and uptake (Sen and Costa, 1986). However, prolonged exposure to soluble NiC12 can also produce substantial damage to heterochromatic chromosomal regions. The modulating effect of Mg in counteracting nickel toxicity is more pronounced in heterochromatin than in euchromatin of CHO cells (Conway et al., 1987). The chromosomal damage induced by particulate NiS may also be related to the production of oxidative species during the process of phagocytosis. Pretreatment of CHO cells with the antioxidant vitamin E (~t-tocopherol) significantly reduced the number of chromosome aberrations produced by NiS, but did not reduce the damage induced by NiC12 ( L i n e t al., 1991). This provides further evidence that nickel carcinogenesis may also involve a multitude of effects including those directly produced by ionic nickel and those related to the active oxygen species produced indirectly during the phagocytic process of nickel uptake. The preferential interaction of nickel with heterochromatin appears to be related to several facets of nickel metabolism and chemistry: its uptake and localization (heterochromatin tends to be localized on the periphery of the nucleus where phagocytized nickel is concentrated) and its ability

50

E.T. SNow

to mimic magnesium ions and interact with Mg 2+ binding sites on proteins. The resultant DNA damage may reflect redox cycling of protein-bound Ni 2+ (Klein et al., 1991b; Linet al., 1991). These interactions with heterochromatin have important consequences relating to the induction of cancer by nickel. For example, the nickel-induced transformation of Chinese hamster embryo cells is often accompanied by varying deletions of the long ann of the heterochromatic X chromosome (Conway and Costa, 1989). Recent research in Dr Costa's laboratory has shown that this X chromosome contains a senescence or tumor supressor gene which can be lost or inactivated in some nickel-transformed cell lines (Klein et al., 1991a). Cellular senescence can be restored by transfer of a normal hamster or human X chromosome into the nickel-transformed cells (Klein et al., 1991a). Other aspects of the nickel-magnesium competition which relate to the carcinogenic potential of nickel have been reviewed by Kasprzak et al. (1987a) and include the ability of magnesium to: Decrease the incidence of nickel-induced tumorigenesis in mice and rats, inhibit the uptake of nickel into target tissues in vivo and into the nucleus of the target tissue cells and inhibit the binding of nickel to DNA in vitro. Magnesium carbonate supressed the localized inhibition of NK cells by nickel subsulfide and decreases nickel carcinogenesis in Fischer rats (Kasprzak et aL, 1987b). Magnesium is also antagonistic to nickel-induced inhibition of intercellular communication in NIH 3T3 cells (Miki et al., 1987). Nickel interference with magnesium-dependent protein binding specific for heterochromatic mouse DNA sequences (Imbra et al., 1989) may be related to effects on chromatin structure or gene regulation. Finally, nickel has been found to alter the kinetics of microtubule polymerization and produce dramatic changes in microtubule morphology in nickeltreated 3T3 cells in vitro (Lin and Chou, 1990). This affect was also noted by Swierenga and McLean (1984) and is likely to contribute to the cellular toxicity of nickel. Nickel-induced alterations in protein-DNA binding and inter- and intra-cellular control processes may have profound effects on both carcinogenesis and tumor promotion.

4.4. OTHER METALS: PROTEININHIBITIONAND DNA REPAIR 4.4.1. Beryllium: Cellular Immunity and Nucleic Acid Metabolism In common with nickel and chromium, beryllium has the ability to elicit a strong immune response, especially in the respiratory system (Kriebel et al., 1988; Rossman et al., 1988). It also affects key enzymes involved in nucleotide metabolism and can decrease the fidelity of DNA replication in vitro (L6onard and Lauwerys, 1987; Skilleter, 1984). Thus, its activities resemble many of those attributed to other carcinogenic metals. Nevertheless, because of its extreme toxicity, the carcinogenicity of beryllium is not as well established as that of other metals, such as nickel and chromium. Beryllium exposure by inhalation produces chronic and acute granulomatous disease, while dermal exposure can result in exzematous dermatitis and granulomatous ulcers; all of these conditions are the result of an extreme immune response to the metal (Kriebel et al., 1988; Reeves, 1989). Patients with acute and chronic beryllium disease also have an elevated risk of developing lung cancer (Steenland and Ward, 1991). Several aspects of the cellular and molecular toxicology of beryllium which may be related to its ability to induce carcinogenesis include: Its poor solubility (Cotton and Wilkinson, 1980) and uptake by phagocytosis (Witschi, 1968); its ability to act as a phosphate analog and thus activate or inhibit key cellular enzymes such as cGMP phosphodiesterase (Bigay et al., 1987), alkaline phosphatase (Thomas and Aldridge, 1966), deoxythymidine kinase (Mainigi and Bresnick, 1969) and Na +, K + ATPase (Skilleter, 1984); and its ability to interfere with gene expression (Perry et al., 1982). The effects on gene expression may be related to beryllium-induced inhibition of protein phosphorylation (Williams and Skilleter, 1983). Possibly as a result of its ability to mimic phosphates, beryllium can also decrease the fidelity of DNA replication in vitro (Luke et al., 1975; Miyaki et al., 1977; Sirover and Loeb, 1976c) and has been shown to induce GC ~ AT transition mutations in E. eoli (Zakour and Glickman, 1984). It has been suggested that the mutagenic activity of Be is due to inhibition of the 3' ~ 5' phosphatase (proofreading) activity of DNA polymerase (Luke et al., 1975).


51

Although it is generally negative in most other short term genotoxicity tests (Ashby et al., 1990; Simmon et al., 1979), beryllium has been reported to induce morphological transformation of mammalian cells in culture (DiPaolo and Casto, 1979; Dunkel et al., 1981; Pienta et al., 1977) and to produce an increase in lung tumors in strain A mice (Ashby et al., 1990). Beryllium alone does not produce chromosomal aberrations, but X-rays plus Be produces a greater than additive response in CHO cells (Brooks et al., 1989). Thus, Be like other metals may inhibit the repair of DNA damage and act in a cooperative manner to enhance the genotoxicity of other agents. The effects of beryllium on cellular metabolism and DNA replication combined with its ability to promote an immune response with concomitant induction of ROS (and oxidative DNA damage to the target tissue) may be sufficient to promote carcinogenesis in vivo. 4.4.2. Cadmium: Protein Interactions and Gene Expression Cadmium is widely used in industry in plating, batteries, plastics and semiconductors. Although the toxicity of cadmium is high and human exposure has been plentiful, the human carcinogenicity of cadmium is not certain because exposure to cadmium is seldom found in the absence of other carcinogenic metals. Evidence suggests, however, that cadmium may be associated with cancer of the lung and prostate in humans (Piscator, 1981; Waalkes and Oberd6rster, 1990). Animal models of cadmium carcinogenesis are well established (for a recent review see Waalkes and Oberd6rster, 1990). In rats, cadmium exposure by subcutaneous injection gives rise to tumors at the site of injection, also in the kidney and in the testes. The induction of testicular interstitial cell tumors correlates with extreme toxicity of cadmium to the testes (Waalkes and Oberd6rster, 1990). However, low doses of cadmium, below those which give substantial tissue damage and tumorigenesis to the testes, can give rise to tumors of the prostate. Inhalation exposure to cadmium aerosols also gives rise to lung tumors (reviewed in Waalkes and Oberd6rster, 1990). Cadmiuminduced tumorigenesis in animals can be reduced by pretreatment with zinc (Waalkes et al., 1989) or a low dose of cadmium; both of which which elicit the production of increased levels of metallothionein. Although cadmium bound to metallothionein (cadmium-thionein) has been reported to be able to cause DNA strand-breaks in vitro (Miiller et al., 1991), increased production of metallothionein in vivo is protective against cadmium toxicity (Coogan et al., 1992a,b), despite its increased cellular uptake. Chronic zinc deficiency increases the toxicity and tumorigenesis of cadmium (Waalkes et al., 1991). Several aspects of cadmium related to its genotoxic potential will be briefly reviewed, including: Effects of cadmium on cell metabolism, gene expression and enzyme activities; competitive antagonism of cadmium-induced genotoxicity by zinc; cadmium-induced DNA damage and repair; and mutagenesis by cadmium. Cadmium is a 'soft' metal; it binds preferentially to sulfhydryl groups in proteins and to DNA bases more than to DNA phosphates (Jacobson and Turner, 1980). Cd resides just below zinc in the periodic table and the two elements share many similar chemical properties. Cadmium also has the same charge and ionic radius as calcium and has been found to alter calcium homeostasis in vivo, even at low environmentally relevant exposures (Staessen et al., 1991). Cadmium is readily taken up by cells and the kinetics of uptake are dependent on cell type. Resistance to cadmium can be induced in bacteria (Rossman et al., 1992). However the most important mechanism for resistance to cadmium and other heavy metals in mammals is the inducible metal-binding protein, metallothionein (for review see Waalkes and Goering, 1990). Lack of metallothionein is one of the reasons that the testes and prostate are a target organs for Cd tumorigenesis (Waalkes and Perantoni, 1986, 1989). Glutathione can also provide some degree of resistance to cadmium by binding the metal (Wahba et al., 1990). The cellular toxicity of cadmium is mediated by cadmium-DNA binding, by inhibition of sulfhydryl-containing proteins and, at least in some instances, by the induction of active oxygen species. Cadmium has been shown to produce lipid peroxidation in rat hepatocytes in vitro (Fariss, 1991) and promote an oxidative burst by pulmonary alveolar macrophages in culture (Zhong et al., 1990). Chromosomal aberrations and DNA strand breaks induced by cadmium can also be reduced by catalase (which inactivates hydrogen peroxide) and mannitol (a scavanger of hydroxyl radicals) (Ochi and Ohsawa, 1985; Snyder, 1988).

52

E.T. SNOW

4.4.2.1. Molecular toxicology o f cadmium. Cadmium genotoxicity is complex and related to its interactions with both DNA and proteins. Cadmium has been shown to bind to DNA in vitro (Jacobson and Turner, 1980; Koizumi and Waalkes, 1990; Waalkes and Poirier, 1984) and can produce protein-associated alkali-labile lesions in the DNA of cultured cells (Coogan et al., 1992b; Nocentini, 1987; Ochi et al., 1983; Snyder, 1988). This damage is at least partially repairable (Ochi et al., 1983; Snyder, 1988). There are at least two types of cadmium-DNA binding sites and metals such as Zn, Mg and Ca which can antagonize cadmium-induced tumorigenesis are competitive antagonists of the high affinity binding (Waalkes and Poirier, 1984). These high affinity cadmium-DNA binding sites involve metal interactions with both the DNA bases and the phosphates and are filled cooperativly by low concentrations of cadmium. The interaction of cadmium with the DNA from rat testicular interstitial cells in vitro can be inhibited by pretreatment of the rats in vivo with zinc (Koizumi and Waalkes, 1990), suggesting that the zinc which remains bound to the DNA through purification can inhibit the binding of additional cadmium. This result is surprising and implies that at least some of the genotoxic effects of cadmium may be due to direct DNA binding of the metal. DNA replication is also inhibited by low concentrations of the metal and the fidelity of both DNA and RNA synthesis is decreased in vitro (Niyogi and Feldman, 1981; Niyogi et al., 1981; Sirover and Loeb, 1976a). It is not known whether this is due to cadmium binding to the DNA or to the polymerase. However, although cadmium-inhibited replication by purified DNA polymerase fl is reversed by the addition of thiols, but not zinc; the effects of Cd on cellular DNA replication and repair are restored by Zn (Nocentini, 1987). Mutagenesis by cadmium has been reported in both bacterial (Mandel and Ryser, 1987) and mammalian cells (Ochi and Ohsawa, 1983; for review see Rossman et al., 1992). Although these results have not been consistently reproduced, recent modifications of the Salmonella mutagenesis protocol have also shown low, but significant, mutagenesis by cadmium (Pagano and Zeiger, 1992). The most consistent finding with regard to cadmium mutagenesis is its ability to enhance the mutagenic potential of alkylating agents. This is because cadmium (and mercury) efficiently inhibit the repair of mutagenic alkylated base adducts, especially O6-alkylguanine. The primary protein which repairs this lesion is the highly conserved, O6-aklylguanine-DNA-alkyl transferase (AGT). As described earlier (see discussion on arsenic), this protein contains at least one essential cysteine residue which accepts the methyl group from the DNA, restoring the original DNA sequence. Cadmium not only inhibits the human (Bhattacharyya et al., 1988), rat and E. coli (Scicchitano and Pegg, 1987) proteins, both cadmium and mercury also inhibit the transcription of the gene and damage-related induction of gene expression (the 'adaptive response' of E. coli) (Suzuki et al., 1991; Takahashi et al., 1988, 1991). Thus, cadmium enhances the mutagenesis (but not toxicity) of alkylating agents in bacteria (Mandel and Ryser, 1984; Takahashi et al., 1991) and also increases carcinogenesis by dimethylnitrosamine in rats (Wade et al., 1987). Cadmium has also been reported to inhibit the repair of u.v.-induced DNA damage (Snyder et al., 1989) and to act as a co-mutagen with u.v. in Chinese hamster V79 cells (Hartwig and Beyersmann, 1989a). Cadmium may also affect cell metabolism and genotoxicity by inducing gene expression. Metallothionein induction acts as a mechanism to reduce cadmium toxicity and can be used as a measure of cadmium exposure (Cosma et al., 1991). Cadmium can in addition induce the expression of other proteins, including: Heat shock proteins (Courgeon et al., 1984; VanBogelen et al., 1987), oxidative stress response genes and heme oxygenase (Keyse and Tyrrell, 1989; VanBogelen et al., 1987), a family of proteins called the acute-phase reactants (Yiangou et al., 1991) and gadd153, a DNA damage recognition protein (Luethy and Holbrook, 1992). The effect of cadmium on gene expression and its interactions with DNA binding proteins such as gadd 153 may be related to its ability to mimic zinc. Zinc plays an important role in many DNA binding proteins through its ability to stabilize the protein fold known as the 'zinc-finger', or 'finger-loop', motif (Berg, 1986). This motif, made up of a highly conserved sequence of appropriately-spaced cysteine and histidine residues, has been found in a great many DNA binding and regulatory proteins, including numerous protooncogenes (Berg, 1986; Sunderman, 1990). It has been found that cadmium can substitute for zinc in these proteins in vitro and it has been postulated that this substitution may


53

TABLE 1. Mechanisms of Genotoxicity Induced by Carcinogenic Metals Metal Compounds Mechanism

Arsenic

Chromium

Nickel

Beryllium

Cadmium

D N A damage D N A binding DPCs Strand breaks Oxidative damage

no ? no? ?

yes yes yes yes

yes yes yes yes

yes? ? no? ?

yes no? yes yes

D N A replication Altered fidelity

yes

yes

yes

yes

yes

D N A repair inhibition Alkyl transferasc

yes?

no

no

?

yes

Excision repair

yes

yes

yes

yes?

yes?

Mutagenesis Bacterial Mammalian Co-mutagenesis

no no yes

yes yes ?

no yes yes

weak weak ?

no yes yes

Gene expression G-ene induction Gone amplification

yes yes

yes no?

? no?

? ?

yes no?

Cellular metabolism Oxidative stress Mg/Ca homeostasis Enzyme inhibition

yes no? yes

yes no? yes

yes? yes ?

no? no? yes

yes yes yes

Cytogenetic effects Chromosome Abs

yes

yes

yes

?

yes

Cellular immunity

no

yes

yes

yes

yes

Cell transformation In vitro

yes

yes

yes

yes

yes

The results presented here represent a compilation of the data discussed in detail in the text. A question mark (?) represents lack of data or, in conjunction with a YES or NO response, conflicting or insufficient data. play role in cadmium carcinogenesis in vivo (Sunderman, 1990). The role of these protein interactions in cadmium genotoxicity, if any, is not known. Thus the mechanisms of cadmium genotoxicity are multifaceted and encompass several of the same processes induced by several other carcinogenic metal compounds discussed in this review; phagocytosis and induction of oxidative bursts by alveolar macrophages, D N A damage, inhibition o f D N A repair and alteration of D N A replication fidelity and the induction of gene expression. Tissue specificity is related, at least in part, to the presence or inducibility of metallothionein. Cadmium carcinogenesis is probably the result of multiple, simultaneous and interrelated events.

5. S U M M A R Y Several different metal compounds have been identified as human carcinogens; however, the mechanisms of carcinogenesis by metals are not well understood. Of the established metal carcinogens, only chromium(VI) compounds are positive in most short term genotoxicity tests. Nickel compounds, although clastogenic and positive in many mammalian mutagenesis assays, are uniformly negative in bacterial systems. Arsenic produces chromosomal damage in mammalian cells and increases the mutagenic response to other agents in both bacteria and cultured mammalian cells, but is not mutagenic by itself. Other metals which have been shown to be carcinogenic in animal models, but have not been proven to be human carcinogens, include beryllium and cadmium. These metals also give weak positive responses in some, but not all, short term assays. As described in detail in the text and summarized in Table 1, these carcinogenic metal compounds

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appear to act by multiple mechanisms to produce genotoxic effects either alone or by enhancing the effects of other agents. Several of the metals, especially nickel, cadmium, beryllium and, to a lesser extent, chromium, produce a strong cellular immune response in vivo. At least some of the genotoxic damage elicited by these metals is due to reactive oxygen species produced during phagocytosis. Phagocytosis of metal compounds by lung macrophages and PMNs produces a cellular response very similar to that produced by classic tumor promoters such as phorbol esters. Other effects of these metals, such as alterations in intercellular communication and gene expression, are also more closely related to tumor promotion than initiation. Most of the metals bind to D N A or inhibit the repair of D N A damage; they all decrease the fidelity of D N A replication in vitro. Each o f these metals also has effects on protein interactions. For example, arsenic and beryllium inhibit enzymes required for oxidative phosphorylation and nucleotide metabolism, respectively. Nickel and chromium induce the formation of D N A crosslinks with chromatin proteins. Cadmium can alter gene expression and enzyme function by binding to essential cysteine residues. Metals such as chromium and, probably, nickel and cadmium act as both initiators and promoters of carcinogenesis and are therefore, by definition, complete carcinogens. The induction o f tumorigenesis by each of these metals is due to the combination of these processes; taken separately they are probably insufficient to produce the multiple genetic changes necessary to initiate and eventually promote the carcinogenic process. Metal carcinogens and possibly all complete carcinogens, are multifaceted and complex--as is the endproduct, cancer. Acknowledgements--The author gratefully acknowledges the many helpful discussions and careful reading of

the manuscript by Drs Catherine B. Klein, Katherine S. Squibb and Jerome J. Solomon. Many thanks also go to Drs Toby G. Rossman, Timothy Coogan and F. William Sunderman, Jr, who kindly sent me preprints of their manuscripts in press. Research of E. T. S. presented here was supported by Grants from the National Institutes of Health: CA45664, ES00260 and CA13343.

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WETTF.RHAHN,K. E., CUP

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