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Molecular and Cellular Mechanisms of Hexavalent Chromium-Induced Lung Cancer: An Updated Perspective A.M. Urbano1,2,3, L.M.R. Ferreira1,2 and M.C. Alpoim1,3,4* 1

Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal; 2Unidade I&D Química-Física Molecular, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Coimbra, Portugal; 3Centro de Investigação em Meio Ambiente, Genética e Oncobiologia (CIMAGO), Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal; 4Centro de Neurociências e Biologia Celular, Coimbra, Portugal Abstract: For over a century, chromium (Cr) has found widespread industrial and commercial use, namely as a pigment, in the production of stainless steel and in chrome plating. The adverse health effects to the skin and respiratory tract of prolonged exposure to Cr have been known or suspected for a long time, but it was much more recently that the toxicity of this element was unequivocally attributed to its hexavalent state. Based on the combined results of extensive epidemiological studies, animal carcinogenicity studies and several types of other relevant data, authoritative regulatory agencies have found sufficient evidence to classify hexavalent chromium [Cr(VI)] compounds as encountered in the chromate production, chromate pigment production and chromium plating industries as carcinogenic to humans. Crucial for the development of novel strategies to prevent, detect and/or treat Cr(VI)-induced cancers is a detailed knowledge of the molecular and cellular mechanisms underlying these pathologies. Unfortunately, in spite of a considerable research effort, crucial facets of these mechanisms remain essentially unknown. This review is intended to provide a concise, integrated and critical perspective of the current state of knowledge concerning multiple aspects of Cr(VI) carcinogenesis. It will present recent theories of Cr(VI)-induced carcinogenesis and will include aspects not traditionally covered in other reviews, such as the possible involvement of the energy metabolism in this process. A brief discussion on the models that have been used in the studies of Cr(VI)-induced carcinogenicity will also be included, due to the impact of this parameter on the relevance of the results obtained.

Keywords: Chromate, carcinogenesis, bronchial. INTRODUCTION 1. The Chemistry of Chromium in Brief Chromium (Cr) is the twenty first most abundant element of the Earth’s crust [1], where it is found mostly in the chromite ore, in combination with iron and oxygen (FeCr2O4) [2]. Curiously, it was in the less prevalent mineral crocoite (PbCrO4) that this metal was discovered, in 1797, by Louis Vauquelin. The term chromium, derived from chroma, the Greek word for “color”, reflects the ability of this element to form strongly colored compounds [3]. Cr can exist in a wide variety of oxidation states, but only the most stable ones, i.e., the trivalent and hexavalent states (Cr(III) and Cr(VI), respectively), occur naturally in the environment in any relevant amounts. All known Cr(III) complexes exhibit an octahedral geometry and, with a few exceptions, are kinetically inert (halflives of several hours) [2,3]. In biological systems, Cr(III) readily reacts with a wide diversity of biological molecules, ranging from small molecules to RNA, DNA and proteins, which may result in interference with their normal functions (sections 8 and 13). In aqueous solution, Cr(VI) exists as an oxyanion, either in the chromate (CrO42–) or in the dichromate (Cr2O72–) form [4]. Which form exists depends strongly on pH and concentration, with the chromate ion predominating under physiological conditions. They both exhibit tetrahedral structures, but differ somehow in their reactivity [2]. The chemical and physical properties of Cr(VI) compounds differ significantly from those of Cr(III) compounds, with important biological consequences. For instance, contrary to Cr(III), Cr(VI) does not interact with macromolecules (section 8). Also, due to a lower mobility, Cr(III) compounds are less bioavailable than those *Address correspondence to this author at the Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Apartado 3126, 3001-401Coimbra, Portugal; Tel: + 351 239 853600/603; Fax:+ 351 239 853607; E-mails: [email protected]; [email protected]

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of Cr(VI). This reflects not only to the lower water solubility of Cr(III) compounds, but also the fact that whereas Cr(VI) forms anionic species, such as CrO42-, Cr(III) forms primarily positively charged compounds, such as [Cr(OH)]2+, which adsorb easily to negatively charged clay surfaces [5]. 2. Adverse Health Effects of Chromium: The Importance of the Oxidation State The adverse health effects of chromium compounds have been known for more than a century, but it was much more recently that the toxicity of this element was unequivocally attributed to the hexavalent state [6]. Trivalent Cr compounds are poorly absorbed by the gastrointestinal tract and are almost invariably innocuous [7], only becoming toxic, by rather nonspecific mechanisms, when administered in concentrations close to their solubility in aqueous solution [8]. In the late 1980s, Biedermann and Landolph showed that Cr(III) compounds were 1,000-fold less cytotoxic than Cr(VI) compounds in cultured diploid human fibroblasts [9,10]. Interestingly, Cr(III) was shown to potentiate glucose tolerance in rats, acting as the active element of a putative dietary compound, named glucose tolerance factor (GTF) [11]. Later, an oligopeptide containing Cr(III), called low molecular-weight chromium binding substance (LMWCr), was isolated from bovine liver and proved to enhance insulin effects [12]. There is still some debate on the effective role played by Cr(III) in glucose tolerance, with suggestions that Cr(III) is not an essential micronutrient, but rather a pharmacological agent that may allay diabetes and heart conditions through interference with iron absorption [13]. Still, it was recently shown that it does improve glucose and insulin tolerance in diabetic mice through modulation of Insulin Receptor Substrate (IRS) phosphorylation [14]. Promising results have been, indeed, obtained in clinical trials where subjects treated with Cr(III) presented lower glucose and insulin circulating levels after acute oral administration of sucrose [15]. The first suspicions that Cr could induced tumors were based on reports of an abnormally high incidence of lung cancers in Scottish

© 2012 Bentham Science Publishers

An Updated Perspective

chrome pigment workers, dating back to the late XIX century [7]. Based on extensive epidemiological, in vivo and in vitro studies, Cr carcinogenicity was confirmed and specifically assigned to the hexavalent state. Although dermal contact with chromium compounds is usually associated with allergic responses, characterized by eczema and contact dermatitis [16], no significant increase in skin cancer was reported among Cr(VI)-exposed workers [17,18]. Actually, based on the data available up to 1990, the only cancers that the International Agency for Research on Cancer could clearly associate with Cr(VI) exposure were those of the lung and of the sinonasal cavity [6]. A decade later, De Flora dismissed all reports of Cr(VI)induced cancers at sites other than the lower respiratory tract and the sinonasal cavity [19]. However, a new evaluation is clearly needed, in face of an increasing number of reports on a variety of other Cr(VI)-induced cancers, including those of the skin, stomach, brain, kidney, bladder and prostate, as well as malignant lymphoma [17,18,20-28]. An increase in mental, psychoneurotic and personality disorders among all race groups [26], in association with the findings that Cr(VI) exposure results in Cr accumulation in the central nervous system of rodents [27, 29-32], raises the possibility that Cr(VI) is also neurotoxic. The different toxicities of Cr(III) and Cr(VI) have been rationalized in terms of structure: with a tetrahedral structure that resembles those of the phosphate and sulfate ions, the chromate oxyanion is readily transported into the cells via the ubiquitous non-specific anion exchangers [33]. This transport is not possible for the large, octahedral Cr(III) complexes. It is interesting to note that the existence of more specific transporters for phosphate in bacteria limits considerably Cr(VI) uptake [34]. 3. Occupational and Environmental Exposure to Chromium Both Cr(III) and Cr(VI) are used extensively in industry: stainless steel and anodized aluminum resistance to oxidation comes from a protective chromium oxide layer [35], lead chromate (PbCrO4; chrome yellow) and chromic oxide (Cr2O3; chrome green) are still used as paint pigments [7], while other chromium compounds are used in leather tanning [36] and to fix dyes to fabrics [37]. The highest exposures to Cr compounds occur in occupational settings, in the form or airborne fumes, mists and dust. These exposures affect several million workers worldwide [6]. Cr(VI) particles with a diameter ranging from 0.2 to 10 μm, such as those originating from coal combustion and brick production [3], represent the biggest oncogenic risk, as such small sizes warrant ready access to the bronchial epithelium upon inhalation [38]. Nonoccupational exposures are also of concern, due to elevated environmental contamination resulting from the release of Cr(VI) compounds from industrial waste disposal, Portland cement, concrete pavement, cigarette smoke, amongst other sources. Although several strategies are being developed in order to remediate pollution by chromium compounds [39], Cr waste is not easily biodegradable, partly due to extensive cross-linking with organic substrates and to adsorption of Cr(III) to surfaces in soils [3,36]. Cr compounds can also be found naturally in the environment, but to a much lesser extent, due to erosion of chromium-containing rocks and volcanic eruptions. These naturally occurring compounds are mostly in the trivalent form. The gastrointestinal tract and lungs are the major routes of Cr absorption. Fortunately, mammals exhibit a large extracellular capacity to reduce Cr(VI), significantly reducing its carcinogenic potential. Most Cr(VI) ingested is reduced by the gastric juice [40] and then by plasma ascorbate (Asc; vitamin C) [41]. The remnant readily crosses erythrocyte membranes [7] and is intracellularly reduced by a variety of small molecules and, possibly, proteins [4] (section 6). Inhaled Cr(VI) particles are chiefly retained by lung tissue, concentrating at major bifurcations, where they may persist for as much as twenty years [42,43]. Cr(VI) oxyanions slowly re-

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leased from these particles are reduced by Asc, which is present in the lavage fluids of the lungs in very high levels [41]. As this release takes place at the cells' surface, some Cr(VI) can be actively transported into these epithelial cells, escaping extracellular reduction. Currently, there is no antidote to chromium intoxication, and the only suggested treatments are based on ascorbic acid administration and soft metal chelating agents [7]. 4. Some Specificities of Chromate-induced Lung Cancer Chromate exposure is now an undisputed independent risk factor for lung cancer [25,44], notwithstanding the fact that the majority of chromate workers that developed lung cancers were also smokers [25,42,43]. Indeed, chromate lung tumors are found at the sites of chromium accumulation [45]. Moreover, chromate cancers exhibit molecular features very different from those of cancers induced by smoking, particularly microsatellite instability (section 11) and a specific pattern of methylation of tumor suppressor genes. In chromate lung cancers, aberrant methylation was detected in the CpG islands of APC (86%), MGMT (20%), hMLH1 (28%) and p16INK4a (33%). In nonchromate lung cancers, it occurred either at lower frequencies or did not occur at all: APC (44%), p16INK4a (26%) and hMLH1 (0%) [46-48]. Chromate lung cancer also forests a low incidence of mutations in the gene coding for the p53 tumor suppressor protein [48]. Moreover, when observed, their pattern differs from that of common squamous cell lung cancers [48,49]. It was also reported that the most prevalent form of lung cancer among Cr(VI)-exposed workers in Japan and in Texas was squamous cell carcinoma [42,45,50], a subtype of non-small cell lung cancer (NSCLC) that is also closely associated with tobacco smoking [51], whereas in the Slovakian population small cell carcinoma prevails [52]. Rather intriguingly, a recent study revealed that exposure to low levels of Cr(VI) led to a significantly increased risk of lung cancer among non-smokers (i.e., among never smokers and subjects who quit smoking at least 20 years before recruitment), but not among smokers [53]. It is likely that either among the smokers the exposure was below a threshold level or that the effects were too small to be detected. 5. Some Considerations on the Importance of the Model System on the Study of the Mechanisms of Hexavalent Chromium Carcinogenicity In spite of the collective effort of a very large number of research groups, our present knowledge of the exact mechanisms underlying Cr(VI) carcinogenicity is still very limited. It is now clear that, similar to most other biological processes, Cr(VI)induced effects are strongly dependent upon the experimental conditions chosen, which may help to explain the apparently contradictory data found sometimes in the literature. In this section, some of the factors that may have contributed to the generation of contradictory data will be briefly discussed. 5.1. Acellular and Cellular Systems The study of the mechanisms of Cr(VI) carcinogenicity has employed a very large number of experimental systems, ranging from chemical (acellular) systems to animals, all of which present their own advantages and limitations. Due to their relative simplicity, chemical studies were particular well suited to investigate certain aspects of Cr(VI) carcinogenesis, namely the intracellular metabolism of Cr(VI) (section 6) and the interactions of Cr(VI) reduction products with DNA (section 8). Nonetheless, particular care must be exerted when extrapolating results to the complex in vivo situation. That only a few of the several cellular components possessing chromate reductase activity are actually capable of significantly reduce this oxyanion under physiological conditions and/or in the presence of toxicologically relevant Cr(VI) concentrations (section 6) illustrates clearly this point.

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Considering that Cr(VI) induces carcinomas, cell lines derived from bronchial epithelium are potentially more informative, as other cell types are likely to differ in terms of Cr(VI) uptake, intracellular metabolism and/or cellular responses [54]. In particular, the specific phenotypes of malignant cells, namely a strong reliance on glycolysis for energy production [55,56], will likely affect the carcinogenic process being modeled. However, for a variety of reasons (e.g., cost), relatively few studies were carried out using nontumorigenic human bronchial epithelial cells. The number of different culture media used in the study of Cr(VI)-induced carcinogenesis is also remarkably high. Some of these media are supplemented with serum, whilst others are serumfree. Moreover, the absence of Asc from commercial media may have compromised the relevance of some of the results obtained (section 6). Another aspect that deserves further investigation is the extracellular reduction of Cr(VI) by media components, which may alter the Cr(VI) concentrations to which cells are actually exposed [57,58]. When using human lung epithelial cells, LHC-9 is probably a good option, as it does not reduce Cr(VI) per se [59]. 5.2. Exposure Regimen The exposure regimen adopted for a given study can have a strong impact on the cellular mechanisms evoked. In particular, Cr(VI) effects observed with very high concentrations may not be observable with lower ones, and vice-versa. One criticism to the theories advocating a dominant role for reactive oxygen species (ROS) in Cr(VI)-carcinogenesis (section 6) is, precisely, the fact that detection of these species by electron spin resonance (ESR), a technique of low sensitivity, required exposing cells to very high Cr(VI) and/or hydrogen peroxide (H2O2) concentrations [60-71], which were clearly unphysiological [72]. The use of 2,7dichlorofluorescin diacetate (DCF-DA) as a probe to evaluate ROS generation by assessing H2O2 levels poses an additional problem, as this probe is known to react faster with Cr(V) than with H2O2 [73]. The concentration-dependent Cr(VI) effects on the antioxidant defense enzymes glutathione peroxidase, glutathione reductase and catalase [74,75] are another example of the influence of the exposure regimen on the results obtained. The prominent antioxidant activities of the glutathione-dependent system observed for low Cr(VI) concentration (2 M) and for the early stages of higher Cr(VI) exposures (>5 M) contrasted with the toxic “pro-oxidant” effect observed upon more prolonged exposures to higher Cr(VI) concentrations (20–30 M). In the latter situation, a marked decrease on the activities of the glutathione-dependent antioxidant system, particularly the glutathione peroxidase/reductase defensive axis, and a concomitant increase in ROS levels were observed. Kim and collaborators also reported increased ROS generation upon acute exposures to very high Cr(VI) concentrations ( 800 μM) [76]. This strongly implies an intracellular threshold that affects the response of the cell to Cr(VI) [77]. As further discussed in section 9, high levels of Cr-DNA binding inhibit polymerase activity [78], which may trigger apoptosis. On the contrary, low levels of Cr-DNA binding increase polymerase activity and processivity, compromising the fidelity of DNA replication [78]. 5.3. The Development of Cellular Models of Cr(VI)carcinogenesis In many aspects, our present knowledge of Cr(VI)-induced lung carcinogenesis has now reached a stage that demands experimental systems that reflect more closely the events that take place in the occupational setting. Animal models are expensive and, in any case, the difficult access to the tracheobronchial tree seriously limits close observation and sequential tissue sampling. Therefore, attempts were made to mimic the in vivo process by inducing the neoplastic transformation of different cell lines by long-term chronic exposure to Cr(VI). To this purpose, primary cultures were

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not an option, as they have extremely short lifespans in culture, precluding mechanistic investigations. Early attempts involved human diploid foreskin fibroblasts, and succeeded in the induction of phenotypic characteristics suggestive of neoplastic transformation, namely anchorage-independent growth [9,10]. Lead chromate induced morphological and neoplastic transformation of cultured C3H/10T1/2 mouse embryo cells [79]. Attempts to induce neoplastic transformation in human bronchial cells are much more recent. In one of these attempts, induction of foci formation and loss of contact inhibition on both fibroblasts and human bronchial epithelial cells was achieved upon a five-day exposure to 0.1–10 μg/cm2 particulate lead chromate [80,81]. In another study, Azad and collaborators induced loss of contact inhibition, colony formation and increased rates of cell invasion, migration and proliferation by exposing nonmalignant human bronchial epithelial cells (BEAS-2B) for 24 passages to 5 M Cr(VI) [82]. Using also BEAS-2B cells, Alpoim and collaborators achieved confirmed neoplastic transformation by chronic exposure (12 passages) to a lower Cr(VI) concentration (1 M), followed by low cell density cultivation [83]. 6. The Intracellular Metabolism of Hexavalent Chromium In aqueous solution and at pH 7.4, the redox potential for the global three electron reduction of Cr(VI) to the very stable Cr(III) (equation 1) is +0,52 V. In biological systems, chromate is even a stronger oxidizing agent, as the formation of Cr(III) is further favored by its binding to the molecules involved in the reduction process. The reduction process can take place at different sites within the cell, such as the cytosol, the nucleus, the mitochondria and the endoplasmic reticulum. It is a fast, stepwise process that originates the very unstable intermediates Cr(V) and/or Cr(IV). These intermediates are probably involved in the formation of the above-mentioned Cr(III) coordination complexes as, contrary to Cr(III), they may be labile to substitution [4]. CrO42– + 4H2O + 3e– Cr(OH)3 + 5OH– (1) The plethora of cellular constituents that can potentially reduce Cr(VI) has been extensively reviewed elsewhere [4,84] and includes Asc, reduced glutathione (GSH), cysteine (Cys), hydrogen peroxide, diols, nicotinamide coenzymes (NADH and NADPH), flavoenzymes and -hydroxycarboxylic acids. There is also a small number of redox proteins that possess chromate reductase activity, namely the heme proteins hemoglobin, cytochrome P450 and complex I of the electron transport chain (ETC) [85,86]. However, the reduction of Cr(VI) must not be evaluated solely from thermodynamic considerations, as can be fully appreciated in its reduction by isocitrate, which, though thermodynamically favorable, is too slow to have any impact on metabolism. Compartmentation is also of significance: as only a small fraction of Cr(VI) partitions to mitochondria [87], the actual contribution of complex I of ETC to the overall Cr(VI) reduction may not be relevant. It now seems well established that the intracellular reduction of Cr(VI) is mostly a nonenzymatic process involving Asc, GSH and Cys, with Asc as the predominant reducer in the target tissues of chromate toxicity [88]. Whereas the intracellular levels of Asc and GSH are both in the millimolar range [89], Asc is a faster reducer. Cys presents a lower intracellular concentration and, apparently, is the slowest reducer. Thus, the reduction of Cr(VI) by Cys only assumes biological significance upon depletion of the other reducers, such as in individuals subjected to prolonged Cr(VI) exposure, which was shown to result in GHS depletion [90]. It is of note that Asc is generally barely detectable in cultured cells. Serum is the only source of this metabolite in most culture media and the low Asc levels provided by serum are rapidly depleted (after just one day in culture, in the case of H460 human lung epithelial cells) [9193]. Under these conditions, GSH and Cys become the main Cr(VI) reducers, which may distort Cr(VI) metabolism and give rise to abnormal responses [93].


Intracellularly, two pathways of Cr(VI) reduction are possible, one via one-electron transfers, sequentially generating Cr(V), Cr(IV) and Cr(III), while the other one is initiated by a two-electron transfer, directly generating Cr(IV). For Asc:Cr(VI) ratios up to 1, the one-electron transfer seems to predominate [94,95]. Above equimolar Asc:Cr(VI) ratios, Asc acts essentially as a two-electron donor, and the generation of Cr(V) rapidly decreases [96-101]. With the thiols GSH and Cys as the reducers, the two pathways of Cr(VI) reduction are also possible [102-104]. Still, kinetic studies have revealed that, at neutral pH and in the presence of physiological Cys levels, the one-electron transfer accounts for over 90% of the reduction [105]. Which of the two pathways predominates in vivo depends also on the specific coordinated ligands and on the presence of other oxidants (e.g., carbohydrates) and catalytic metals such as Fe, due to adventitious Fenton reactions [106-109]. The Cr intermediates Cr(V) and Cr(IV) are not the only very reactive species formed during the intracellular reduction of Cr(VI). The concomitant oxidation of the reducing agents generates a wide range of carbon and sulfur reactive species, such as Asc-derived carbon-centered alkyl radicals and formyl radicals [63,110,111]. Moreover, extensive ESR studies by the group of Shi revealed that, in the presence of H2O2 or lipid peroxides, Cr(VI) reduction by certain reducers generated not only long-lived Cr(V) species, but also the very reactive hydroxyl radical (.OH) [63,106,110,112-114]. Using the same spectroscopic technique, several authors confirmed the generation of Cr(V) and hydroxyl radicals, following the reduction of Cr(VI) by microsomes, mitochondria and vitamin B2 [6062,115-118]. Fenton-like reactions (equations 2 and 3) were proposed for the generation of the hydroxyl radical. Cr(IV) + H2O2 Cr(V) + .OH + OH (2) (3) Cr(V) + H2O2 Cr(VI) + .OH + OH Following these early results, two theories emerged proposing a dominant role for oxidative DNA damage in Cr(VI)-carcinogenesis: Shi and Dalal advocated a central role for ROS [111,119], while Kawanishi and collaborators presented a “tetraperoxochromate(V)” theory of carcinogenesis [120]. However, as previously discussed (section 5.2), the use of the extremely high Cr(VI) and H2O2 concentrations required for ESR detection of these paramagnetic species may have compromised the validity of these theories. Notwithstanding, in vivo reports revealed, somehow consistently, ROS association with liver and kidney injuries observed in rodents exposed to soluble Cr(VI) compounds [29] and with overexpression of matrix metalloprotease-9 (MMP-9) in lung lesions induced by zinc chromate in Big Blue rats [121]. MMP-9 is a zinc-dependent endopeptidase that is activated by ROS and plays an important role in cell invasion and metastasis [122]. Other authors prefer to emphasize the role of Cr-DNA adducts in Cr(VI)-induced carcinogenesis as, contrary to oxidative DNA damage, this type of lesion can be observed even under relatively mild Cr(VI) exposure conditions [101,123,124] (section 8). 7. Redox Imbalance Produced by Hexavalent Chromium Abnormalities in aerobic respiration can lead to overproduction of ROS, mainly at mitochondria [125], which exert dose-, time- and cell type-dependent actions. These range from DNA, membrane and protein damage [126] to the enhancement of specific signaling pathways, apoptosis [127] and activation of heat shock proteins [128]. To protect themselves against the deleterious actions of ROS, cells maintain adequate levels of specific reduced molecules (e.g., NADPH and GSH), which scavenge these reactive species. Enzymes such as glutathione reductase, which depends on NADPH for its function [129], and thioredoxin, an intracellular protein responsible for keeping other proteins in their reduced state [130], are also important antioxidant defenses. Cellular antioxidant defenses may be compromised by an extensive Cr(VI) reduction, but more data is needed to confirm a po-


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tential correlation. Analysis of lymphocytes from stainless steel welders revealed a significantly lower glutathione content [89], but, rather surprisingly, Cr(VI) concentrations as high as 400 μM did not diminish the intracellular pools of reduced glutathione in bronchial epithelial cells [131]. In isolated mitochondria, Cr(VI) treatments in the micromolar range depleted the NADH pool, an effect that was attributed to inhibition of some mitochondrial dehydrogenases and to NADH oxidation by Cr species (it was demonstrated that intermediate Cr(V) was capable of oxidizing an equimolar amount of NADH in a few seconds) [86]. However, as mentioned before, Cr(VI) partitions very moderately to the mitochondria [87]. In BEAS-2B cells, both the cytosolic (Trx1) and the mitochondrial (Trx2) forms of thioredoxin participated directly and significantly in Cr(VI) reduction: 10 μM of sodium chromate oxidized ca. 20% of Trx2 in three hours, while 25 μM of the same salt yielded a partial oxidation (50%) of Trx1 within the same time. In Cr(VI)-treated erythrocytes, glutathione reductase activity was conspicuously decreased [132]. Asc rescued its activity, while it had no effect in the absence of Cr(VI) [132], emphasizing the importance of Asc in vivo and partly explaining its utilization as a treatment for chromium poisoning [7]. The occurrence of redox reactions between glutathione reductase and Cr(VI) has recently been confirmed [131]. 8. Cr(VI)-induced DNA Lesions Although the interaction of Cr with nucleic acids is likely to play an important role in Cr(VI)-induced carcinogenesis, in vitro studies have clearly demonstrated the inability of Cr(VI) to bind or otherwise interact with these macromolecules [133,134]. On the contrary, several of the species generated during the intracellular reduction of Cr(VI), most notably Cr(III), bind to them. In this sense, Cr(VI) is frequently regarded as a pro-carcinogen. Cr(III) can coordinate DNA either directly or via the intermediate Cr(IV) and Cr(V) [135]. As Cr(III) tends to establish coordinated bonds with its intracellular reducers, Cr-DNA adducts in mammalian cells are mostly in the form of ternary complexes [135]. Cys-Cr(III)-DNA and GSH-Cr(III)-DNA were the most abundant Cr(III)-DNA ternary complexes detected under conditions of Asc deficiency [136], whereas Asc-Cr(III)-DNA predominated in the presence of physiological levels of Asc [101,135]. The intracellular metabolism of Cr(VI) induces many other types of DNA lesions, either due to direct Cr-DNA interaction or as a result of oxidative damage. These include DNA-protein crosslinks (DPCs), DNA inter/intrastrand crosslinks (ICLs), single- and double-strand breaks (SSBs and DSBs, respectively), oxidized bases and abasic sites [84,137]. The relative amounts of Cr(III), Cr(IV) and Cr(V) might be a major factor determining the DNA damaging activity of Cr(VI) [138]. For instance, whereas Cr(III) plays a critical role in the formation of DPCs, Cr(V) does not, at least in human lung adenocarcinoma cells (A549) [139]. As to ICLs, they could be detected in the in vitro reduction of Cr(VI) by Asc [140,142], or Cys [141, 143], but not by GSH [141]. Their formation was highly dependent on the ratio of reducer to Cr(VI), the most extensive DNA crosslinking occurring under conditions of limited reducer concentrations [137]. It has been proposed that oxidative DNA damage is mainly caused by the intermediate oxidation states of Cr, occurring either by direct electron abstraction [107,109], via the formation of Cr(V)peroxo intermediates [108,120] or via ROS action [106,107,119]. This type of DNA damage is presumably formed locally in lung tissue only [144] and just under certain occupational exposure conditions, such as those observed in chrome pigment production, where workers can exhibit Cr blood levels up to 5 M [145,146]. SSBs can have a primordial role in genomic instability in rapidly dividing cells, such as cancer cells, as they can collapse replication forks during DNA replication, originating the highly genotoxic DSBs [147]. These can trigger cell cycle arrest and eventually


apoptosis via activation of p53 [148-150]. The presence of SSBs in the livers and kidneys of mice intraperitoneally exposed to Cr(VI) was used as an indicator of a generalized oxidative insult on DNA [137,151]. In fact, oxidized bases can give rise to abasic sites, followed by strand scission. Moreover, it was proposed that strand breaks can occur as a result of hydrogen abstraction from the ribose moiety by hydroxyl radicals generated during the reduction of Cr(VI) by GSH [152,153]. These assumptions are supported by the observed inhibition of Cr(VI)-mediated SSB formation, in normal human bronchial epithelial cells (HBE), upon addition of catalase and iron chelators, which avoided ROS generation by Fe-induced Fenton reaction [67]. A similar result was obtained upon increase of GSH levels [151]. In isolated DNA systems, the induction of DNA strand breaks in the presence of Cr(VI) and GSH was also shown to be ROS-dependent [154]. However, stabilized Cr(V) species such as bis-(2-ethyl-2-hydroxy-butanato)oxochromate(V) were also able to bind directly to phosphate groups in isolated single-stranded and double-stranded DNA and abstract hydrogen atoms from either the C1 or C5 position of the ribose moiety [155,156]. In cDNA microarray analysis of normal human lung cells treated with toxicologically relevant concentrations of Cr(VI), no clear evidence for the involvement of ROS on this type of lesion could be found [137]. As discussed later in this review (section 14.1.1), SSBs can also result from repair of primary DNA lesions. 9. Cr(VI) Effects on Replication and Transcription Early cellular studies revealed that both the formation of Cr(III)-deoxyribonucleotides (dNTP) complexes [157] and the direct oxidation of dNTPs [158] reduced the dNTP pool, provoking a sharp decrease in DNA biosynthesis [58]. Additionally, some of the genetic lesions induced by Cr(VI), namely DPCs and ICLs, represent physical obstacles for the replication and transcription processes [123,159,160]. Importantly, in vitro experiments performed with Cr(III) and single-stranded DNA (ssDNA) demonstrated that different doses of Cr may lead to radically different outcomes: when at concentrations low enough to generate only 3 or 4 Cr-DNA adducts per 1000 nucleotides, polymerase processivity and speed were, rather surprisingly, increased by several fold. At higher concentrations, ICLs formed and the usual replication blockade was observed [78]. The higher rate of DNA synthesis was accompanied by a diminution in replication fidelity, which may, at least in part, explain the mutagenicity of Cr(VI) compounds (section 10). It is of note that polymerase-based misincorporation assays, although very expedite in assessing replication blockade, do not include several proteins required for DNA synthesis and repair in mammals and, as such, are not always representative of the in vivo situation [161]. Still, studies using intact cells have confirmed that Asc-Cr(III)DNA ternary adducts halt DNA replication [137]. Unsurprisingly, Cr-induced stereochemical changes in the DNA molecule also affect transcription, mainly at the RNA chain elongation stage [162], suggesting that the lower protein synthesis rate after chromate treatment observed in early studies [58] was provoked by lower levels of mRNAs. Importantly, high mobility group (HMG) proteins, which recruit transcription factors, were found to bind to Cr-DNA in a dose-dependent manner, presenting a 2.5-fold preference for DNA isolated from tissues treated with 10 μM potassium dichromate over normal ones [163]. 10. The Mutagenicity of Cr(VI) In studies carried out in the early 1980s in subcellular systems, both Cr(III) and Cr(VI) compounds decreased the fidelity of E. coli DNA polymerase I [164], but only the latter were mutagenic in bacterial and mammalian cell systems [165]. Later studies confirmed the occurrence of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, as well as chromosomal aberrations, in Cr(VI)-exposed hamster cells [61,62]. Biedermann and Landolph demonstrated that many Cr(VI) compounds induced

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mutations to 6-thioguanine resistance in cultured primary diploid human foreskin fibroblasts [9,10]. Binary adducts are only weakly mutagenic [101,123] and the degree of mutagenicity of the ternary complexes formed between Cr, DNA and Cr(VI) reducers varies according to the reducer [91,101,123]. Asc-Cr(III)-DNA complexes appear to be the most mutagenic, as Cr(VI) concentrations that were completely unmutagenic under Asc-deficient conditions became mutagenic in the presence of physiological Asc levels [101,135,166]. When present at 2 mM, GSH generates the weakly mutagenic GSH-Cr(III)-DNA adducts on pSP189 plasmids. Increasing GSH levels up to 5 mM resulted in approximately 4-times greater DNA adduct-normalized yield of mutations, caused by nonoxidative mechanisms [167]. In terms of animal models, it was reported that the intratracheal instillation of soluble potassium dichromate in Big Blue transgenic mice induced a time- and dose-dependent increase in the frequency of mutations at doses above 3 mg/kg. Depletion of tissue GSH with buthionine sulfoximine before Cr(VI) treatment led to a decrease in the frequency of mutations [168]. 11. Cr(VI)-induced Genomic Instability Genomic instability (GI) is present in the vast majority of human cancers and has recently been classified as an emerging hallmark of cancer [169]. GI can occur in the form of gross chromosomal abnormalities (i.e., chromosomal instability) and/or small-scale genetic changes (e.g., microsatellite instability). As mentioned before (section 4), the majority of Cr-related lung squamous cell carcinomas analyzed are characterized by microsatellite instability [46]. Rather surprisingly, no microsatellite instability could be detected when BEAS-2B cells were neoplastically transformed by Cr(VI). Persistent aneuploidy was observed, though [83]. It has also been shown that exposure to both Cr(III) [170] and Cr(VI) [171] induces the formation of micronuclei, i.e., chromosome fragments that fail to be comported by the nucleus during cell division and which can be seen as a hallmark of chromosomal instability in cancer [172]. This clastogenic action of Cr(VI) compounds is consistent with their ability to generate DNA strand breaks (section 8). 12. The Carcinogenicity of Cr(VI) Compounds: The Influence of Solubility and Administration Route Attempts to induce tumors in animals upon Cr(VI) exposure led to results that, although not totally consistent, do point to the importance of both solubility and administration route in determining the outcome. Soluble Cr(VI) compounds induced very few, if any, lung tumors, and only upon inhalation [173-178]. Conversely, Cr(VI) compounds of sparing aqueous solubility were able to evoke a carcinogenic response independently of the exposure route [174,179186]. The contrasting results of soluble versus particulate Cr(VI) compounds can be explained in terms of their relative inactivation via extracellular reduction. As mentioned before (Section 3), mammals exhibit a large extracellular reducing capacity, rapidly converting most Cr(VI) oxyanions to Cr(III). Therefore, the only chromate ions that can escape extracellular reduction and be transported into epithelial cells are those that are slowly and continuously released from particulate compounds that adhered to the surface of those cells [187-192]. Many of those failed attempts to induce tumors provided useful data. When Sprague-Dawley rats were instilled for 30 months with sodium dichromate, apoptosis was increased in both bronchial epithelium and lung parenchyma, concomitant with an increased expression of 13 out of 18 apoptosis-related genes [177]. The authors, therefore, proposed apoptosis as a likely protective mechanism at a post-genotoxic stage of Cr(VI) carcinogenesis. In another study, involving the intrabronchial exposure of rats to soluble Cr(VI), it was possible to detect the induction of squamous metaplasia in the bronchial epithelium, a transformed state from which squamous carcinomas may arise [186]. Interestingly, a persistent


carcinomas may arise [186]. Interestingly, a persistent lung inflammatory response was found in several of the studies involving the exposure of rodents to soluble Cr(VI). This process is closely related to cancer and genetically regulated by the same gene loci [175,186,193-196]. Finally, in a very recent work attempting at monitoring, in BALB/c mice, the different stages of lung tumor formation upon intranasal infiltrations with highly insoluble zinc chromate particulates, degenerative changes in the proximal and midproximal bronchiolar mucosa and sloughing of epithelial cells were observed [121, 197]. The injuries were centrally located in the lung, whereas nearby pleural regions remained uninvolved. 13. Chromium-protein Interactions and their Consequences There are several examples in the literature of Cr interaction with proteins. As these interactions interfere with protein function, ultimately altering cellular processes, they are worth exploring in the context of Cr(VI)-induced carcinogenesis. Already in the 1970s, Schoental hypothesized that, in cells exposed to Cr(VI) compounds, epoxyaldehydes derived from hydrolysed tissue lipids could participate in the cross-linking of proteins through their amine and sulfidryl groups [37]. Kinetic studies have also demonstrated that small polynuclear hydroxo Cr(III) complexes inhibit bacterial colagenases [36], which is in line with what has been long known in the tanning industry, i.e., that Cr(III) helps preventing deterioration of dead skin [198]. Cr(VI) can also act as an enzyme cofactor, as already described, in the 1940s, by Stickland, who assessed the effects of a plethora of metal ions on the activity of phosphoglucomutase (PGM), an enzyme involved in the metabolism of glycogen [199]. Although Mg2+ was always indispensable for full activity of this enzyme, 0.1 mM Cr(III) was able to impart some activity. Notably, at lowerthan-normal concentrations of Mg2+, only Cr(III) could impart maximal activity to PGM, when present at ca. 10 μM. Noteworthy, a recent chemical genomic screening has identified a PGM inhibitor as a potent anti-proliferative drug for breast cancer [200]. On the other hand, Cr(III) forms complexes with ATP (Cr-ATP) that resemble Mg-ATP complexes (the major form of intracellular ATP [201] and can behave, essentially, as competitive inhibitors for a variety of proteins, such as 3-phosphoglycerate, pyruvate kinase [157,202] and the Na+/K+ ATPase [203]. Some mitochondrial dehydrogenases are also affected by chromate. -Ketoglutarate dehydrogenase is particularly sensitive (3–5 μM Cr(VI) for 50% inhibition) [86]. This potent inhibition may have a mechanism similar to that of Cd2+ action: the metal ion binds to the dithiol group of the lipoyl moiety of the enzyme (just as with thioredoxin) [131], even when an electron acceptor alternative to NAD+ is used [204]. This could also be the basis for the chromate inhibition of pyruvate dehydrogenase [86], as these two enzymes have this moiety in common [204,205]. One of the well defined phenotypes of cancer cells is a strong dependence on lactic fermentation for energy generation, which requires an increased glycolytic flux [55,206] (section 16). Unfortunately, with the exception of a preliminary broad study with glycolytic and pentose phosphate pathway enzymes [132] that revealed no significant differences in activity after Cr(III) and Cr(VI) treatment, no literature reports on Cr effects of chromium on glycolytic enzymes could be found. Notwithstanding, Cr(VI) does provoke oxidative damage in several of these enzymes [207]. Also, as Cr(III) can form inert complexes with ATP, it is a potential inhibitor of kinases involved in this pathway [157,202] and of ADP and GDP phosphorylation [158]. 14. Molecular and Cellular Events Downstream of Cr(VI)induced Genotoxicity To maintain genome fidelity, cells possess a refined set of surveillance and regulatory mechanisms, termed cell cycle checkpoints, which ensures that, during each cell cycle, DNA replication


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and chromosomal segregation are completed in an orderly manner [208]. These checkpoints, which are controlled by a complex and highly organized signal transduction network, detect damaged DNA, coordinate cell cycle progression with DNA repair and/or activate pathways that trigger apoptosis. Moderate levels of DNA damage result in transient cell cycle checkpoint arrest, necessary for DNA repair. If complete repair is achieved, cells can regain their replicative potential. Otherwise, cells should permanently exit the cell cycle, either by terminal growth arrest or by apoptosis. Elimination or perturbation of these checkpoint mechanisms may result in the clonogenic survival of cells with damaged DNA, hence in the genesis of cancer. Short-term studies have confirmed that, depending on the extent of the DNA damage, Cr(VI) treatment of lung cells can elicit the three above-mentioned cellular responses [93,140,166,209-220]. Cellular responses to Cr(VI) exposure also include dysfunctional DNA replication and transcription, as well as deregulated DNA repair and survival pathways [77,84]. The precise mechanisms that control cellular responses to Cr(VI) exposure remain poorly understood. Reports of intracellular ROS generation upon Cr(VI) exposure suggested a typical oxidative stress response. Namely, Azad and collaborators argued that Cr(VI)-induced apoptosis in human lung epithelial H460 cells was specifically mediated by the superoxide anion, which mediated the Cr(VI)-induced degradation of the antiapoptotic protein Bcl-2 via the ubiquitination pathway [221]. More recently, the same group observed that reactive nitric oxide, NO, participated in the process that conveyed resistance to apoptosis and impelled Cr(VI)-exposed BEAS-2B cells to malignancy. During the process, that lasted 24 passages in the presence of 5 M Cr(VI), low basal levels of superoxide anion were also detected [82]. These results, although undoubtedly important, must be carefully analyzed as the Cr(VI) concentration (20 M) used to evaluate NO presence was rather different from the one used to drive cells to malignancy (5 M). 14.1. Repair of Cr(VI)-induced DNA Lesions: Mechanisms and Potential Side-effects As DNA repair systems are lesion-specific, several of them are likely activated in response to the wide range of DNA lesions induced by Cr(VI). Deficiencies in these repair systems can be associated with both the onset and the progression of cancer, as an increased frequency of mutations can result in the activation of oncogenes and inactivation of tumor suppressor genes. Although the number of studies concerning the repair mechanisms involved in the removal of Cr(VI)-induced DNA lesions is still limited, they have already yielded some very pertinent results, some of which form the basis of a new theory of Cr(VI)-induced carcinogenesis (section 14.1.3). 14.1.1. The BER/AP Repair Systems Putative Cr(VI)-induced oxidative DNA damage likely activates base excision repair (BER) and the apurinic/apyrimidinic (AP) endonuclease repair system. BER recognizes damaged (oxidized or alkylated) bases and excises them through the use of specific DNA glycosylases, such as 8-oxo-guanine DNA glycosylase 1 (OGG1). This excision results in the temporary formation of AP sites. AP endonucleases (APE1 in humans and APN1/2 in yeast) recognize these AP sites and make an incision in the phosphodiester backbone 5 to the abasic site, generating SSBs containing a 5deoxyribophosphate [222]. These SSBs are subsequently recognized by a DNA repair complex containing DNA polymerase (Pol ) and the X-ray cross-complementing group 1 (XRCC1)-DNA ligase III (XRCC1-Lig III) complex. Pol removes the 5deoxyribophosphate and inserts the correct deoxyribonucleotide, whilst XRCC1-Lig III complex seals the remaining nick in the DNA backbone. In humans, somatic mutations and loss of heterozygosity in OGG1 have been associated with lung tumors [223-225]. Interest-


ingly, exposure of A549 cells to Cr(VI) concentrations (>25 M) highly favorable to ROS generation and, consequently, to 8-oxo-2deoxyguanosine formation inhibited the expression of OGG1, both at the mRNA and protein levels [144,226]. This might have increased the susceptibility of the cells to Cr(VI)-induced mutations, as was observed in APN-deficient yeast [227]. Nevertheless, the absence of cell death suggests that other DNA repair pathways were involved in the removal of the Cr(VI)-DNA lesions. When Kim and colleagues performed a similar study using a different culture medium and employing other methodologies to evaluate OGG1 expression and oxidative DNA damage, results could not be reproduced [76], which may have resulted from differences in the reductive activation of Cr(VI), in the induction of DNA damage and/or in DNA repair. In recent studies with CHO cells, chemical inhibition of BER actually decreased Cr(VI)-induced mutagenesis [228]. Since cells were exposed to much lower Cr(VI) concentrations (< 6 M), DNA lesions other than oxidized bases may have been generated. Overall, these results suggested that: (i) the repair of Cr lesions by BER is possibly error-prone; (ii) in the absence of a functional BER/APE axis, due to BER deficiency, other DNA repair pathways will be involved [228]. Studies on the role of XRCC1 in the repair Cr(VI)-induced SSBs involved CHO and CHO-derived, XRCC1-deficient EM9 cell lines. While XRCC1 deficiency did not affect cytotoxicity by soluble Cr(VI) compounds, it had a strong effect in the case of particulate compounds [229,230]. The fact that the fagocytic internalization of lead chromate particles is much more active in EM9 cells than in the parental CHO cells may account for this effect [187,189,190]. These studies also revealed that XRCC1 protects cells from lead chromate-induced chromosome instability (CIN), as deficiency in this protein resulted in a dramatic increase in the number of chromatid exchanges, a common feature of lung cancer cells [231,232], including those of Cr(VI)-induced cancers [233]. The levels of isochromatid lesions remained unchanged. 14.1.2. Nucleotide Excision Repair Contrary to the small-sized binary Cr-DNA monoadducts, the bulky ternary Cr-DNA complexes, such ICLs and DPCs, may perturb the DNA helical structure [234]. Nucleotide excision repair (NER) is considered the major repair mechanism for these adducts in human cells [235,236]. This system excises a fragment of the damaged strand containing the lesion, followed by repair synthesis, using the intact strand as a template [237-239]. In mutant CHO cells, NER deficiency by loss of either xeroderma pigmentosum complementation group D (XPD) (5# to 3# helicase) or xeroderma pigmentosum complementation group F (XPF) (5#-endonuclease) resulted in impaired removal of Cr-DNA adducts and increased sensitivity to Cr(VI) lethality [236]. NER-deficient XPA, XPC and XPF human lung fibroblasts were also severely compromised in their ability to repair Cr(III)-DNA lesions [235]. It is of note that, in both the yeast S. cerevisiae [228] and in prokaryotes [240], removal of DNA lesions by NER was found to be error susceptible, possibly due to error-prone ligation and/or repair synthesis. More recently, it was also shown that NER-deficient CHO cells exhibited attenuated mutagenesis (monitored at the chromosomal locus hgprt) and lack of clastogenic effects [228], suggesting that, in the absence of a functional NER, damage may be either channeled into another, more precise, error-free repair pathway into error-free bypass of the damage, i.e., error-free translesion synthesis (TLS) [241]. 14.1.3. Mismatch Repair The genetic stability of human cells is strongly dependent on mismatch repair (MMR) system [242], as it corrects single base mispairs and insertion/deletion errors arising during DNA replication homologous recombination (HR), base oxidation and methylation and other biological processes [243,244]. This repair system participates in the cytotoxic responses to several chemotherapeutic drugs, including SN1-type methylating agents [245,246], cisplatin

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[247,248] and halogenated nucleotides [249-251], as well as in the responses to Cr(VI) [93,252]. Loss or defects in this repair system are associated with highly elevated rates of spontaneous mutagenesis, genome-wide instability (it is a cause of MSI found in 15% to 20% of human cancers), predisposition to certain types of cancer, resistance to chemotherapeutic agents, as well as with abnormalities in meiosis and sterility in mammalian systems [242,253]. The loss of MLH1 expression [46], an important protein of the MMR repair system [254,255], and a high incidence (>80%) of MSI [233] are, precisely, two important features of chromate-lung cancers. MMR activity requires an initial recognition of and binding to the damaged DNA region, which is achieved by two heterodimeric complexes: MutS (MSH2-MSH6) and MutS (MSH2-MSH3) [256,257]. MMR activity also requires replication protein A (RPA) and the 5 to 3 double-strand hydrolytic activity of exonuclease 1 (Exo1) [258,259]. These four activities support a mismatchprovoked excision reaction directed by a 5 strand break which terminates upon mismatch removal. After binding to the damaged region, the complex MutS/ recruits the complex MutL (a MLH1-PMS2 heterodimer), resulting in MutS-MutL complex activation and the subsequent excision of up to 1 kb of newly synthesized DNA [260,261]. It has been reported that MMR-dependent processing of DNA damage induced by alkylating agents leads to the production, after the first S phase, of persistent single-strand gaps, and that the subsequent collapse of replication forks leads to a delayed formation of DSBs, occurring in the second S phase [262,263]. Asc-Cr-DNA ternary adducts can be seen as high affinity substrates for MMR proteins, as their replication frequently generates base mispairs. Hence, Cr(VI) exposure can also induce the formation of DSBs by a MMR-dependent mechanism, as confirmed by the groups of Patierno and Zhitkovich [166,264-266]. Using lung epithelial cells (H460 and HBE) and lung fibroblasts (IMR90) exposed to low Cr(VI) concentrations (0.2–2 M) in the presence of physiologic Asc concentrations, the group of Zhitkovich confirmed a direct connection between MMR proteins (MSH2 and MLH1) and DSB formation [166,265]. DSB formation was quantitatively evaluated by p53 binding protein 1 (53BP1) foci formation and -H2AX expression, as well as by micronuclei formation [267-270]. However, contrary to the delayed generation of DSBs induced by alkylating agents, DSB generation by Cr(VI) was observed soon after exposure to this carcinogen [166,252,265], suggesting a distinct mechanism of formation. The fact that XRCC1-deficient (EM9) and proficient (wild-type) CHO cells survived equally to low-moderate Cr(VI) concentrations [151] implies that the collapse of replication forks that may have, ultimately, generated the DSBs did not result from SSB formation, but rather from Asc-generated bulky blocking lesions (e.g. Asc-Cr-DNA) [166,265]. These studies evidenced that Cr(VI)-induced DSB production had a unique requirement for MSH3 and that the MSH2-MSH3 heterodimer acted downstream of MSH2-MSH6. The fact that the -H2AX focus-containing cells were positive for cyclin B1 suggests that Cr(VI)-induced MSH6/3 foci accumulation and DSB formation did not require replication, but only the progression of cells through late S into early G2 phase. This would create the acceptable conditions for cleavage of both DNA strands [265] at low and moderate Cr(VI) concentrations [166,264,265]. As explained by the authors, this may be due either to the induction of Exo1 in late S phase [259] and/or because S phase cells display higher thresholds for checkpoint activation than G2 cells [271]. DSB formation can also result from hairpins or loops in single-stranded regions containing repetitive sequences. Yet another mechanism of DSB formation cannot, at this stage, be excluded, as the transition of cells with single-strand breaks from S to G2 could inactivate the mechanisms of fork viability resulting in collapsed forks and, consequently, in DSB formation [166,265]. DSBs are the most dangerous DNA lesions, as progression of G2 cells with unrepaired DSBs into mitosis generates chromosome


rearrangements and, as a consequence, genomic instability. Therefore, their presence elicits normally a strong apoptotic response [272], which can explain the MMR-dependent mechanism of apoptosis observed in Cr(VI)-treated cells described by the group of Zhitkovich [252]. Having confirmed that the absence of a functional MMR led to decreased Cr(VI)-induced DSB and micronuclei formation, this group proposed that Cr(VI) carcinogenesis may result from the selection, upon prolonged exposure to Cr(VI), of Cr(VI)-resistant, MMR-deficient cells carrying cancer promoting mutations [166,252,273]. The fact that the neoplastic transformation of BEAS-2B cells generated a malignant cell line, RenG2, with a functional MMR system and without MSI [83] is in apparent contradiction with this theory. These conflicting results may have resulted from: (i) the use of different cell lines (although both of human respiratory tract origin); (ii) the fact that BEAS-2B cells are SV40 immortalized and, as such, have a nonoperational p53 pathway [274]; (iii) the use of different culture media and exposure regimens. According to a very recent study, the helicase/exonuclease DNA repair Werner syndrome protein (WRN), which facilitates repair of stalled and collapsed replication forks [275], translocates to -H2AX nucleoplasmic foci and is involved in the repair of Cr(VI)-induced DSBs generated during the S phase of the cell cycle. Accordingly, human cells deficient in WRN protein are hypersensitive to Cr(VI) toxicity and exhibit a delayed reduction in DSBs and stalled replication forks. 14.2. The Development of Resistance to Cr(VI) Cytotoxicity Chromate workers often exhibit lung tissue injury and, in some cases, perforation of the nasal septum and/or respiratory tract ulcerations [6]. Considerable data support a role of apoptosis in the remodeling of lung tissue after acute lung injury [276-279]. Interestingly, it has been observed that lung tissue that has undergone cellular turnover in response to recurrent cytotoxic Cr(VI) exposure may present an attenuated response to subsequent exposures [53]. Reports of an increased resistance to apoptosis and/or necrosis upon continuous exposure of human lung fibroblasts (HLF) and epithelial cells (BEAS-2B) to Cr(VI) [213,220] support this hypothesis. This increased resistance to cell death might contribute to lung cancer development upon chronic exposure to Cr(VI). 14.3. Signaling Pathways Mediating the Cellular Responses to Cr(VI) There is a clear understanding that the disclosure of the signaling pathways that are affected by Cr(VI) downstream of its genotoxic effects, namely those that regulate cell survival and proliferation, is a key issue to understand the molecular events leading to Cr(VI)-induced carcinogenesis. Gene expression profiles are excellent tools to seek and dissect those pathways. Once again, the use of different animals/cell lines and different exposure regimens may evoke different responses, as they will both critically influence the dynamic balance of intermediates formed during the reductive metabolism of Cr(VI) and, as a consequence, determine structural changes in DNA and in protein motifs that bind DNA. 14.3.1. Cr(VI) Effects on Gene Expression The studies on the effects of Cr(VI) on gene expression conducted so far have unraveled a strong dependency on the cell line and exposure regimen employed. For instance, whereas exposure of BEAS-2B cells for 4 h to 10 M Cr(VI) downregulated the expression of 44 genes (corresponding to 90% of the genes analyzed), including C-MYC, CYCLIN K, CYP1B1, MAPKNPK-2, PP1A, FGFR1, HSP90 and AKT1 [280], exposure of A549 cells to 300 M Cr(VI) for 2 h increased the expression of 150 genes and reduced the expression of another 70 [281]. Two time-course studies carried out recently, employing different cell lines and exposure regimens, have both revealed a transient and selective regulation of gene expression by Cr(VI), albeit with a different pattern [83,282]. In one of them, involving A549 cells exposed for 24 h to 10 M


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Cr(VI) [282], a decrease in the expression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (Her2/ErbB2) was observed within the first 4 h, contrasting with an increased expression of ErbB2 and the return to the basal levels of EGFR at 24 h. In the other, in which BEAS-2B cells were exposed for 12 weeks to 1 M Cr(VI), the expression of several genes, including c-MYC, HIF-1, LDH-A, EGFR, DNMT1, cyclins CCND1 and CCNB1, the mitogen-activated protein (MAP) kinases JNK, ERK and p38, and the proteins involved in DNA repair MLH1, RAD51, XRCC5, XRCC1, XRCC3 and OGG1, varied randomly [83]. These cells were subsequently cultured at very low density, again in the presence of Cr(VI), giving rise to malignant subclonal cell lines. Gene expression analysis of these malignant cell lines showed a consistent increase, along time in culture, of all genes analyzed, including the above-mentioned oncogenes (c-MYC, EGFR, HIF-1 and LDH-A) [83]. The observed up-regulation of MLH1 in this in vitro cell system contrasted with the absence of MLH1 expression and microsatellite instability in chromate cancers [46,233]. Studies in normal human lung fibroblasts exposed to Cr(VI) showed that p16 expression remained unaltered [283], again in contrast with what was observed in chromate lung cancers [48]. Studies on the evaluation of Cr(VI) effects on oxidative stressrelated genes (catalase, glutathione S-transferase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxidase, NAD(P)H:quinone oxidoreductase, heme oxygenase 1 (HMOX1) and interleukin 8) in lung cell lines that differed in their transformation status (normal human lung LL 24 cells, human lung adenocarcinoma A549 cells and SV40-immortalized BEAS-2B cells) revealed opposite effects for HMOX1, glutathione peroxidase and Cu/Zn-superoxide dismutase [280,281,284,285]. Discrepant effects on HMOX1 gene expression were also observed in lung cells of C57BL/6 mice intranasally exposed to Cr(VI) [285]. Concerning protein expression, opposing signaling effects were reported for normal cells and transformed cells lacking functional signal transducer and activator of transcription 1 (STAT1). For instance, Cr(VI) stabilized HIF-1 protein and induced the activation of Sp1 as well as of vascular endothelial growth factor A (VEGFA) in cancer cells [286,287], while it had opposite effects on BEAS-2B cells [288]. Cr(VI) can induce gene silencing both genetically (via DNA adducts and ROS) [137,235,287,289] and epigenetically (through alteration of transcriptional complexes) [285,290-292]. The selective activation of kinase cascades, suggested to play an important role in the etiology of Cr(VI)-induced pulmonary diseases, may explain Cr(VI) effects on transcription factors and gene transactivation [293]. 14.3.2. Cr(VI) Effects on Transcriptional Initiation Gene expression is strongly influenced by the activity of transcription factors and it has been demonstrated that many of them are sensitive to Cr(VI) exposure. For instance, in A549 cells, the transcription factor p53, the most widely known tumor suppressor protein, was reported to respond to Cr(VI) by changing its DNA binding ability [294,295]. In addition to p53, Cr(VI)-induced effects on NF-B and several other transcription factors that activate the transcription of genes involved in inflammation, carcinogenesis and pro- or antiapoptotic pathways were studied both in malignant and nonmalignant human respiratory tract cell lines using a wide range of Cr(VI) concentrations. As illustrated in Table 1, while the response of some transcription factors to Cr(VI) was, to a certain extent, independent of the cell model and exposure regimen (e.g., cAMPresponse-element-binding protein (CREB) and AP-1), the response of others, such as HIF-1 and NF-B, was rather erratic and, in certain cases, quite dependent on the experimental conditions used [76,286,287,290,293,296].

cite as 'references cited therein' for HIF 1a and NF kB


Particularly interesting to notice were the effects of Cr(VI) on STAT3 and STAT1 transcription factors, studied in BEAS-2B [293]. Exposure to 5 μM Cr(VI) led to a delayed and prolonged activation of STAT3 and a rapid and short-lived STAT1 activation. This unbalanced activation was suggested to convey profound consequences on cell fate due to the opposed roles of these two transcription factors in lung disease and innate immune system response. STAT1 is a downstream effector of interferon (IFN) signaling [297] and is required to induce innate immune antiviral and antiproliferative responses [298,299]. But, in the absence of STAT1, as observed in a number of cancers [299,300], IFN stimulates STAT3 and STAT5, providing a survival advantage and leading, possibly, to neoplastic transformation [299,301,302]. Moreover, in the airways, loss of STAT1 is recognized to increase the severity of viral lung infections [303] and metal- or chemicalinduced injury [304-306]. In BEAS-2B cells, one major consequence of Cr(VI)-induced STAT1 phosphorylation and nuclear translocation was the reported inhibition of VEGFA expression [288], as this growth factor, in lung epithelial cell models, elicits anti-apoptotic responses [307,308] and is activated following hypoxia [309] and other insults that stabilize HIF-1 [310]. Notably, in respiratory tract cells lacking STAT1, Cr(VI) stimulates both HIF-1 and Sp1 transactivation, as well as VEGFA expression [288]. However, in BEAS-2B cells, HIF-1 was, apparently, unaffected by Cr(VI) exposure [288,306] suggesting that Cr(VI) has opposite signaling effects in normal and in transformed cells that lack functional STAT1. As a matter of fact, in normal cells, Cr(VI) would limit inducibility of protective genes or genes involved in injury repair while, in transformed cells, Cr(VI) might promote proliferation and tumor growth by increasing VEGFA or other growth factors expression [288,306]. Thus, the use of cells whose functional STAT1 signaling status is questionable and different culture conditions stressed, once again, the need for appropriate cell models and exposure regimens. Barchowski’s group work markedly suggests that prolonged STAT3 and transient STAT1 activations, mediated by the Src family kinases Lck and Fyn, respectively, and the consequent accumulation of the inflammatory cytokine interleukine 6 [293,306], may have important repercursions on lung cancer onset, as chronic inflammation is implicated in the development of several cancers, including lung cancer [311] (section 15). 14.3.3. The Role of Epigenetic Mechanisms on Transcription Factors Effects Cr(VI)-induced DNA-protein cross-links, which are found preferentially in nuclear matrix DNA, where many replication, repair and transcription proteins associate, were proposed to play an important role in the epigenetic events associated with Cr(VI) exposure [312]. For instance, Cr(VI) cross-linking of histone deacetylase-1 (HDAC-1) with DNA methytransferase 1 and chromatin was reported to repress, in rat hepatoma cells, the aryl hydrocarbon receptor (AHR) mediated transactivation of CYP1A1 [292,313, 314]. The binding of Cr, by preventing the release of HDAC-1 from chromatin and the recruitment of p300, inhibited the transactivation of CYP1A1 and the induction of over 50 different genes involved in a variety of signaling transduction pathways [314]. Similarly, Cr(VI) blocked TNF--induced NF-B-dependent gene transactivation [290,315], possibly because Cr obstructed the binding of the p65 subunit to CBP/p300, an essential step for NF-B-enhanced transcriptional activity [290]. Even though histone deacetylation is often correlated with gene repression, HDAC-1 forms complexes with STAT1-containing interferon-stimulated gene factor 3 and facilitates the recruitment of RNA polymerase II to IFN-stimulated gene promoters [316,317]. Therefore, Cr(VI) cross-linking of HDAC-1 to chromatin may be the central event leading to the repression of AHR-mediated transactivation of CYP1A1 in rat hepatoma cells [292,314] and the in-

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duction of innate immune gene promoter IRF7 in BEAS-2B cells [306]. Still, it is evident that, in addition to its direct action on chromatin and DNA [314,318], extra-nuclear signaling events are necessary for Cr(VI) to stimulate both the formation of the transactivation complexes and gene repressors [306]. Cr(VI) also attenuated As(III)-induced HMOX1 expression, both in vivo and in BEAS-2B cells [285]. Although Cr(VI) effects occurred in the enhancer region containing critical antioxidant response elements (ARE), the mechanism is different from the one observed with AHR-mediated transactivation of CYP1A1, which was reported to depend on the presence of promoter-proximal sequences [314]. In the case of As(III), the mechanism for Cr(VI)attenuated transactivation was suggested to involve the reduction of the nuclear levels of the transcription factor Nrf2, and As(III)stimulated Nrf2 transcriptional complex binding to the ARE cis element [285]. High Cr(VI) levels (100 M) were also reported to disrupt the transactivation activity of metal transcription factor-1 (MTF-1), in cells other than respiratory tract cells [291]. The study revealed that Cr(VI) shifted the patterns of co-activator interactions, by interfering with the function of all MTF-1 different transactivation domains, i.e., the acidic, the proline-rich and the serine-threonine-rich domains [291]. These data contrast with the long-standing belief that Cr(VI) inhibits the transactivation of inducible genes [290,292,314,318], even though it fails to affect constitutive gene expression. Indeed, the expression of housekeeping genes, such as -actin or albumin, was not affected by Cr(VI) exposure [289,289,319], perhaps, as suggested [320], because the less compact chromatin structure of inducible promoters makes them better targets for Cr binding than the more compact chromatin of constitutive promoters. 14.3.4. The Involvement of p53 in the Signaling Pathways Activated by Cr(VI) The tumor suppressor protein p53 is involved in many pathways that are activated in response to DNA damage, namely cell cycle arrest and apoptosis, and is a key player in neoplastic transformation [148,321]. Studies in bronchoalveolar cells revealed that Cr(VI)-induced apoptosis was p53-dependent and was achieved by effective proapoptotic proteins, PUMA and NOXA. However, p53dependent cell cycle arrest could not be observed [218]. A less known mechanism of Cr(VI)-induced apoptosis operates in a p53independent manner. Hayashi and colleagues used a lymphoma cell line in order to gain insight into this process [322]. ROS generated during intracellular reduction of Cr(VI) produce lipid peroxidation, DNA damage and a decrease in the mitochondrial membrane potential. These events ultimately lead to DNA fragmentation and apoptosis via an increase of cytosolic Ca2+ levels and caspase-3 activity. The fact that ROS can induce a wide variety of modifications in DNA and in signal transduction proteins [67,323,324] tremendously increases the number of intracellular targets that may be activated downstream of a Cr(VI) insult. Gene expression profile studies have, indeed, confirmed the broad effects that Cr(VI) can have at this level [217]. 14.3.5. Activation of Protein Kinases One of the most relevant signaling pathways as far as cancer is concerned is the MAP kinase cascade, as it allows cells to tightly regulate their mitosis and division in response to mitogens [325]. Actually, deregulated protein tyrosine phosphorylation is responsible for the maintenance of proliferative signals and is involved in the early stages of neoplasia [149]. There are over a dozen mammalian MAP kinase genes. The best known belong to the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK(1-3)) and p38 (alpha, beta, gamma and delta) families [326]. Prolonged exposure to Cr(VI) concentrations as low as 0.3 μM led to activation of ERK2 in H4 rat hepatoma cells. At 3 μM, both


Table 1.


293

Cr(VI) Effects on Transcriptions Factor Expression and DNA Binding Ability

Transcription Factors AP-1

CREB

Cell Lines

[Cr(VI] M

BEAS-2B

Consequences

ROS

References

5

Increased DNA binding

[293]

MDA-Human breast carcinoma

2, 20, 100


[287]

H411E Rat hepatoma

2, 20, 100


[287]

Activation

[290]

A549

5

BEAS-2B

10

Increased gene expression

[280]

BEAS-2B

5


[293]

BEAS-2B

5


[293]

Increased expression

[286]

c-Jun binding to CREB

GAS/ISRE HIF-1

NF-B

DU145 human prostate

0.65-10


2, 20, 100


[287]

H411E Rat hepatoma

2, 20, 100

No effect on DNA binding

[287]

BEAS-2B

5


[288]

BEAS-2B

1.25-10


[296]

A549

> 12.5

A549

5-20


[290]

A549

5-100

Increased p53 accumulation and DNA binding

[295]

Inhibition of apoptosis

p53


+

[76]

Apoptosis Increased p53 nuclear accumulation

300

+

[294]

SIE

BEAS-2B

5


[293]

Sp1

BEAS-2B

1.25-10


[296]


2, 20, 100


[287]

H411E Rat hepatoma

2, 20, 100


[287]


[293]

VEGFA inhibition

[306]


[293]

Fyn –mediated STAT1 rapid, short lived activation Lck –mediated STAT3 prolonged activation

BEAS-2B

HBE -Primary human bronchiolar epithelial

5

5

IL-6 production 5

BEAS-2B STAT4 and STAT5

BEAS-2B

ERK1 and ERK2 were activated. These effect relied on the generation of peroxides upon Cr(VI) reduction and was abolished by a significant increase of intracellular GSH levels [327]. In A549 cells, 10 M Cr(VI) selectively activated JNK by specific Src family kinases, namely Fyn and LcK, through a thiol-dependent pathway. Although a concomitant increase in ROS levels was observed, it was demonstrated that ROS generation was not required for JNK activation [328]. In this and several other cell lines, higher Cr(VI) levels activated JNK, ERK and p38, either individually [286,295] or simultaneously [76,327,329]. In spite of yielding a tyrosine protein phosphorylation pattern similar to that observed with protein kinase C (PKC) inducers [330], Cr(VI)-induced activation of MAP kinases was found to be PKC-independent and not subject to down-regulation [327]. These


[306]

IL-6 production 5

No nuclear translocation

[293]

inducers are tumor promoters that activate PKC uninterruptedly and, as a consequence, stimulate constitutively cell growth and division [331]. Interestingly, findings from our laboratory suggest that, in the sub-micromolar range, Cr(VI) induces an enhanced proliferation in human bronchial epithelial cells [220] (Ferreira et al., unpublished results). Akt, a serine/threonine kinase, is an important survival protein that, once phosphorylated, initiates a cascade of signaling events that affect neovascularization, proliferation and apoptosis [332]. Perturbations of the Akt signaling pathway have been associated with non-small cell lung cancer and with poor patient prognosis [333]. In human lung fibroblasts, exposure to 1 and 2 M Cr(VI), chemically inhibited protein tyrosine phosphatase (PTP), result in protein phosphorylation. This was associated with increased cell


proliferation, clonogenic survival and forward mutations at the hgprt locus [334, 338]. The same group then demonstrated that Akt activation by chemically-induced PTP inhibition rescued cells from Cr(VI)-induced G1/S checkpoint and was necessary for the PTP inhibitor effects on pRb and p27, the key mediators of the G1/S transition [335]. Activation of Plk1, a key regulator of mitosis that presents an aberrant activity in tumors [336], rescued cells from Cr(VI)-induced G2/M checkpoint [337]. These events overrode Cr(VI)-induced effects and enhanced the clonal expansion of genetically compromised cells in response to the deregulation of survival signaling pathways. Conversely, in the absence of PTP inhibition, the involvement of the phosphatidylinositol-3-OH (PI3)/Akt signaling cascade in the effects of 1 M sodium chromate was ruled out [338], in spite of reports that this pathway is involved in the toxic mucosal injury and acute inflammatory response in BALB/c mice exposed to particulate zinc chromate [121,197]. These antagonic findings may be due to significant differences in the respiratory systems of mice and humans, to the lack of cross-talk information with other lung tissue components in cellular studies and/or to inappropriate exposure regimens. Additionally, it was established that a Ras/c-Raf/Mek dependent, but Erk-independent pathway mediated the observed Cr(VI) effects in human lung fibroblasts [338]. Apparently, Erk was not mediating Cr(VI)-induced cytoxicity, even though higher levels of Cr(VI) (6 M) led to its transient activation [283], in accordance to what was observed in other studies [328,339,340]. These findings suggest that, for low Cr(VI) exposures, there is either a novel Erk-independent signaling pathway, mediating Mek downstream signaling, or Mek alone, once activated, translocates to the nucleus and regulates cell growth or interacts with cytosolic effectors that regulate cell survival/growth [338]. Recently, studies with BEAS-2B cells revealed that Cr(VI)treatment inhibits a cascade of events culminating in the activation of VEGFA by nickel (Ni2+). The underlying signaling pathways involve Erk, PI3K and the downstream effector Src kinase (a transactivator of AP-1), leading to the downstream activation of HIF-1, STAT3 and, finally, VEGFA [288]. 14.3.6. Signalling Pathways Activated in Response to the Formation of Double-strand Breaks In response to DSB formation, the P13 kinases ataxiatelangiectasia mutated protein (ATM), ATM-Rad3-related (ATR) and DNA-PK phosphorylate the histone H2AX at Ser-139 [267], generating -H2AX [341-344]. This initiates the phosphorylation of several downstream targets (e.g., p53, BRCA1, CHK2, FANCD2, MDM2, NBS1, Rad17, SMC1), triggering G1/S, G2/M and S phase checkpoints [345]. The 53BPI acts upstream of ATM [346]. In dermal fibroblasts and HeLa cells, Cr(VI)-induced S phase DSB formation activated ATM, ATR and their targets p53, CHK2 and SMC1, triggering S phase checkpoint [215,216,269]. It was also shown that an active ATM was required for recovery from Cr(VI)-induced cellular growth arrest [215]. In SV40-immortalized human fibroblasts and HeLa cells, the Cr(VI)-induced activation of signaling pathways mediated by ATM was shown to depend on the intensity of the insult. Thus, while 10 M Cr(VI) induced the phosphorylation of H2AX and of 53BP1 [264,269,347], 40 M Cr(VI) required the Rad17-ATR-SMC1 pathway to induce S-phase checkpoint activation [269]. In primary human lung epithelial cells, exposure to Cr(VI), DSB formation and the subsequent phosphorylation of H2AX and of 53BP1 occurred in G2 phase [166]. This finding is in conformity with the fact that 53BP1 is required for p53 accumulation, G2-M checkpoint arrest and the intra-S phase checkpoint [348]. The intricate connections of different DNA repair pathways are well evidenced in the interplay between the ATM sensor axis and the HR and Fanconi Anemia (FA) pathways [349-352]. HR can be described as a series of interrelated pathways that intervene in the

Urbano et al.

recovery of stalled or broken replication forks and in the repair of DSBs and ICLs, providing critical support for DNA replication [353]. The key reactions that typify HR are catalyzed by RAD51, a central core of proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3). Activation (monoubiquitination) of the FA complementation group D (FANCD2) protein and its interplay with RAD51 is a major requirement for the activation of the HR pathway [354-356]. FA-A cells are unable to activate FANCD2 [264]. Accordingly, they are hypersensitive to DNA crosslinking agents [357-359] and, consequently, to apoptotic death and clonogenic lethality [360]. These effects were reported to result from DSB formation during the S phase of the cell cycle. Accordingly, when both FA-A skin fibroblasts and normal human skin fibroblasts were exposed to Cr(VI) (1–3 M), DSB formation induced FANCD2 monoubiquitination in normal fibroblasts only. Surprisingly, both cell lines had similar levels of S phase-associated -H2AX expression. This observation led to the assumption that, at least in FA-A cells, DNA repair pathway(s) other than the FA pathway were involved on the repair of Cr(VI)-induced DSBs [361]. This is in line with reports of HR subpathway(s) independent of FANCD2 protein and potentially independent of RAD51 [362]. In CHO cells with intact HR and FA DNA repair capacity, exposure to soluble Cr(VI) was reported to trigger RAD51 foci formation and the induction of HR repair [363], whereas CHO-derived cell lines with compromised DNA repair capacity, due to either XRCC3 or RAD51 deficiency, showed increased sensitivity to both soluble and particulate Cr(VI) compounds and increased number of metaphases with chromosome damage, as well as chromatid exchanges following lead particulate Cr(VI) exposure [364]. These results are in consonance with the findings that, with the reported exposure regimen and cell lines, Cr(VI)-induced DSBs occurred preferentially during DNA replication [264,361] and indicate that several repair pathways cooperate in repairing Cr(VI)-induced DNA damage. Still, no definite conclusions can be taken, since these studies were performed in cells other than human respiratory tract cells and encompassing impaired DNA-repair capacity, including cells deficient in Ku80 (a protein of the non-homologous end joining (NHEJ) repair system), XRCC1 and FANCG, which exhibited large differences in the levels of intracellular Cr(VI) compared to their parental and gene complemented control cells [229,365,366]. Cr(VI)-induced DSBs seem to be preferentially formed after the S phase of the cell cycle. When G2 cells with unrepaired DSBs progress to mitosis, chromatid aberrations and, consequently, chromosome instability are observed. Such is the case of cells deficient in HR repair, as this repair system dominates in S and G2 phases, in contrast with NHEJ, which predominates in G1 phase [367,368]. This may explain why Ku80 does not play a role in protecting cells from lead chromate-induced chromosome damage, while it protects them from Cr(VI) cytotoxicity. Apparently, in CHO-derived cell lines, cytotoxicity is uncoupled from chromosome damage, suggesting either the absence of a sensor for those DNA lesions that drive to chromatid aberrations, or that Cr(VI) acts also as an aneugen, binding to the mitotic spindle and causing chromosome missegregation and genetic instability, similarly to what is observed with asbestos [369]. 15. A Potential Role for the Immune System in Cr(VI)-induced Lung Cancer Many chromate workers develop respiratory diseases, including fibrosis and hyperplasia of the bronchial epithelium [6]. Rodents exposed to different chromates or to chromium trioxide particles also developed chronic lung inflammation, with infiltration of neutrophils and macrophages in the central zone of the lung [121,194, 195,197,370,371]. The presence of lymphoid proliferating cells and binucleated macrophages in mice exposed repetitively to zinc


chromate provided additional evidence that chromate exposure induces, in the lung, a pro-inflammatory microenvironment [121]. These cells are commonly found in lung tissue under certain pathologic conditions [372,373] or upon exposure to carcinogens [374]. Subacute exposures of Wistar rats to 25 and 50 g/m3 of sodium dichromate (28–40 days) resulted in activated alveolar macrophages with stimulated phagocytic activities and significantly elevated antibody responses. Conversely, higher Cr(VI) concentrations resulted in a decreased humoral immune system response [194]. All of these chronic inflammatory situations may predispose to lung cancer [311,375-377]. In fact, several lines of evidence suggest that the progression towards full malignancy of cells that bear potentially oncogenic mutations requires a permissive tissue microenvironment, which can easily be provided by chemokines (ROS and reactive nitrogen species) and cytokines (e.g., interleukins and metalloproteases) secreted by inflammatory cells, particularly macrophages and neutrophils, key players in angiogenesis and metastasis [378]. For instance, individuals with lung inflammation due to chronic infection, asbestos exposure, interstitial lung disease and asthma are at an increased risk for lung cancer [375-377], a risk that decreases with the use of nonsteroidal anti-inflammatory drugs [379,380]. The chemokines and cytokines released create a favorable microenvironment that directly promotes and exacerbates DNA damage and/or interferes with DNA repair mechanisms [68,378,381]. As such, the repeated influx of neutrophils, followed by the chronic presence of macrophages, leads to repetitive tissue injury, thereby promoting the proliferation of cells with a selective growth advantage, which can explain the degenerative changes and sloughing of epithelial cells observed in the proximal and midproximal bronchiolar mucosal injury present in mice airways following exposure to either Cr(VI) alone [121,197] or to Cr(VI) and asbestos [382], and in rodents exposed to welding fumes [383-386]. In response to an injury, neutrophils release proteases that digest the collagens of the extracellular matrix, playing a very important role in inflammation and cancer development [311,387]. Interestingly, increased pro-metalloprotease-9 (MMP9) and IL6 levels were found in the airways of mice following repetitive exposure to zinc chromate [121,197]. MMP-9 was shown to play important roles in other lung diseases and is critical in neutrophilic inflammation [388]. 16. Effects of Hexavalent Chromium on Energy Metabolism The concept of cancer as a metabolic disease emerged in the 1920s, following the seminal studies of Otto Warburg [389]. These studies clearly demonstrated that cancer cells exhibit a specific metabolic phenotype, relying strongly on glycolysis to satisfy their energy needs, even in the presence of ample oxygen. Although this concept was largely ignored for many decades, the metabolic reprogramming of cancer cells has now become an emerging field in cancer research [56]. This was due, in a large manner, to the successful exploitation of the specific metabolic phenotype of cancer cells in the powerful diagnosis technique positron emission tomography (PET) [390] and to its promising potential in cancer therapeutics. All studies dealing with Cr(VI)-induced metabolic reprogramming carried out so far consistently unveiled changes in bioenergetic parameters that are in line with the metabolic shift first described by Warburg. Cr(VI) visibly diminished oxygen consumption rates, both in cultured cells and in isolated mitochondria, which was mainly attributed to the oxidation of NAD-linked substrates (e.g. glutamate, citrate) by Cr(VI) [85,86,391]. Ryberg and Alexander also observed that the inhibitory effect of Cr(VI) on respiration was reduced when isolated mitochondria were incubated with an uncoupler and chromate before the respiration was triggered by addition of glutamate, indicating that Cr(VI) interfered directly with the electron flow through ETC [85].


295

The relevance of nucleosides and their derivatives in macromolecular synthesis, cellular redox state and energy transduction has been long known [392]. Using radioactively labeled nucleosides, Bianchi and co-workers [57] found out that pyrimidine nucleoside uptake was more stimulated by Cr(VI) than purine uptake. In this study, the kinetics of intracellular pool saturation were consistent with an action of Cr(VI) not only at the membrane level (activation of nucleoside permeases), but also on nucleoside metabolism, as saturation level, but not saturation time, was affected. Recent studies with PC-12 cells extended the previous results, as they clearly showed that cells treated with micromolar concentrations of Cr(VI) suffered a shift in energy metabolism. Glucose uptake rates were significantly higher than control values and the adenylate energy charge decreased [393]. This experimental system, which consists in cells living in suspension, resembles the one used by Warburg to ultimately prove that cancer cells have inherently higher glucose consumption rates, even in well aerated environments [55]. More recently, it was observed that chronic exposure of BEAS-2B cells to 0.1 and 1 μM of Cr(VI) led to increased glucose uptake and lactate production (Ferreira et al., unpublished results). Cr(VI) in concentrations as low as 2.5 μM visibly augmented the expression of the transcription factor HIF-1 in prostrate carcinoma cells [286]. HIF-1 is involved in oxygen sensing and is frequently overexpressed in cancers [310]. Considering that HIF-1 affects the expression of numerous genes involved in energy metabolism, its increased expression in Cr(VI)-treated cells may contribute to the metabolic shifts observed. Interestingly, it was found that, in BEAS-2B, Cr(VI) had exactly the opposite effects in HIF-1 expression, also in micromolar concentrations. This discrepancy was due to the presence of STAT1, an important factor during response to injuries in normal airways. This could be confirmed using cells deficient in this protein [288]. ACKNOWLEDGEMENTS MCA and AMU thank Centro de Investigação em Meio Ambiente, Genética e Oncobiologia (CIMAGO; Grants 16/06 and 26/07, respectively). AMU acknowledges also a grant from Fundação para a Ciência e Tecnologia (FCT), Portugal (FCT; Grant PEstOE/QUI/UI0070/2011). LMRF was the recipient of a FCT grant (BII/FCTUC/C2008/QFM/BQ/2ªFASE). Due to space limitations, it was not possible to discuss all of the studies carried out in areas covered by this review, and the authors apologize for these omissions. REFERENCES [1] [2] [3] [4] [5] [6]

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Received: December 27, 2010

Revised: January 3, 2011

Accepted: May 23, 2011

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