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 (FeCr

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Molecular and Cellular Mechanisms of Hexavalent Chromium-Induced Lung Cancer: An Updated Perspective

A.M. Urbano

^1,2,3

, L.M.R. Ferreira

^1,2

and M.C. Alpoim

^1,3,4*

1Departamento 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 fac- ets 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 (FeCr

2

O

4

) [2]. Curiously, it was in the less prevalent mineral crocoite (PbCrO

4

) that this metal was discovered, in 1797, by Louis Vauquelin. The term chromium, de- rived 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 octahe- dral geometry and, with a few exceptions, are kinetically inert (half- lives 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 (CrO

₄^2–

) or in the dichromate (Cr

2

O

7

2–

) 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 reac- tivity [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, Apar- tado 3126, 3001-401Coimbra, Portugal; Tel: + 351 239 853600/603; Fax:+

351 239 853607; E-mails: [email protected];

[email protected]

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 CrO

4

2-

, 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 hex- avalent 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 ad- ministered 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]. Interest- ingly, 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 contain- ing Cr(III), called low molecular-weight chromium binding sub- stance (LMWCr), was isolated from bovine liver and proved to enhance insulin effects [12]. There is still some debate on the effec- tive role played by Cr(III) in glucose tolerance, with suggestions that Cr(III) is not an essential micronutrient, but rather a pharma- cological 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) phosphory- lation [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 su- crose [15].

The first suspicions that Cr could induced tumors were based on reports of an abnormally high incidence of lung cancers in Scottish

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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 Interna- tional 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 personal- ity 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 ration- alized in terms of structure: with a tetrahedral structure that resem- bles 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 exis- tence 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: stain- less steel and anodized aluminum resistance to oxidation comes from a protective chromium oxide layer [35], lead chromate (PbCrO

4

; chrome yellow) and chromic oxide (Cr

2

O

3

; chrome green) are still used as paint pigments [7], while other chromium com- pounds 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 expo- sures affect several million workers worldwide [6]. Cr(VI) particles with a diameter ranging from 0.2 to 10 μm, such as those originat- ing 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 con- tamination 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 ca- pacity 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-

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 re- lease takes place at the cells' surface, some Cr(VI) can be actively transported into these epithelial cells, escaping extracellular reduc- tion. Currently, there is no antidote to chromium intoxication, and the only suggested treatments are based on ascorbic acid admini- stration and soft metal chelating agents [7].

4. Some Specificities of Chromate-induced Lung Cancer

Chromate exposure is now an undisputed independent risk fac- tor for lung cancer [25,44], notwithstanding the fact that the major- ity 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 in- duced 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 carci- noma 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 expo- sure 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 Car- cinogenicity

In spite of the collective effort of a very large number of re- search 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 con- ditions chosen, which may help to explain the apparently contradic- tory data found sometimes in the literature. In this section, some of the factors that may have contributed to the generation of contradic- tory 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 simplic-

ity, chemical studies were particular well suited to investigate cer-

tain aspects of Cr(VI) carcinogenesis, namely the intracellular me-

tabolism of Cr(VI) (section 6) and the interactions of Cr(VI) reduc-

tion 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 pos-

sessing chromate reductase activity are actually capable of signifi-

cantly 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, intra- cellular 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 car- cinogenic process being modeled. However, for a variety of reasons (e.g., cost), relatively few studies were carried out using nontu- morigenic 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 serum- free. 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 proba- bly 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 (H

2

O

2

) concentrations [60-71], which were clearly unphysiological [72]. The use of 2,7- dichlorofluorescin diacetate (DCF-DA) as a probe to evaluate ROS generation by assessing H

2

O

2

levels poses an additional problem, as this probe is known to react faster with Cr(V) than with H

₂

O

₂

[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 expo- sure 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 de- crease 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 bind- ing inhibit polymerase activity [78], which may trigger apoptosis.

On the contrary, low levels of Cr-DNA binding increase polym- erase 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, at- tempts 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

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 neoplas- tic transformation of cultured C3H/10T1/2 mouse embryo cells [79]. Attempts to induce neoplastic transformation in human bron- chial 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/cm

²

particulate lead chromate [80,81]. In another study, Azad and collaborators induced loss of contact inhi- bition, colony formation and increased rates of cell invasion, migra- tion 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 pas- sages) 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 fa- vored 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].

CrO

₄^2–

+ 4H

₂

O + 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 inclu- des 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 com- plex I of the electron transport chain (ETC) [85,86]. However, the reduction of Cr(VI) must not be evaluated solely from thermody- namic 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 mito- chondria [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 reduc-

ers, 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) [91-

93]. 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].

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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 physiologi- cal 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].

con- centrations required for ESR detection of these paramagnetic spe- cies may have compromised the validity of these theories. Notwith- standing, in vivo reports revealed, somehow consistently, ROS as- sociation with liver and kidney injuries observed in rodents exposed to soluble Cr(VI) compounds [29] and with overexpression of ma- trix metalloprotease-9 (MMP-9) in lung lesions induced by zinc chromate in Big Blue rats [121]. MMP-9 is a zinc-dependent en- dopeptidase that is activated by ROS and plays an important role in cell invasion and metastasis [122]. Other authors prefer to empha- size 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. En- zymes such as glutathione reductase, which depends on NADPH for its function [129], and thioredoxin, an intracellular protein re- sponsible for keeping other proteins in their reduced state [130], are also important antioxidant defenses.

Cellular antioxidant defenses may be compromised by an ex- tensive Cr(VI) reduction, but more data is needed to confirm a po-

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 bron- chial epithelial cells [131]. In isolated mitochondria, Cr(VI) treat- ments in the micromolar range depleted the NADH pool, an effect that was attributed to inhibition of some mitochondrial dehydro- genases 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 par- tial oxidation (50%) of Trx1 within the same time. In Cr(VI)-treated erythrocytes, glutathione reductase activity was conspicuously de- creased [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 glu- tathione 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 intermedi- ate Cr(IV) and Cr(V) [135]. As Cr(III) tends to establish coordi- nated 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 in- ter/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 for- mation of DPCs, Cr(V) does not, at least in human lung adenocar- cinoma 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 cross- linking occurring under conditions of limited reducer concentra- tions [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 con- ditions, 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 rap-

idly dividing cells, such as cancer cells, as they can collapse repli-

cation forks during DNA replication, originating the highly geno-

toxic DSBs [147]. These can trigger cell cycle arrest and eventually

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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, fol- lowed 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 mi- croarray analysis of normal human lung cells treated with toxico- logically 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 di- rect 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, repre- sent physical obstacles for the replication and transcription proc- esses [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 con- centrations, 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 elonga- tion stage [162], suggesting that the lower protein synthesis rate after chromate treatment observed in early studies [58] was pro- voked 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 potas- sium 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 con- firmed 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

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 un- mutagenic 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 hu- man cancers and has recently been classified as an emerging hall- mark of cancer [169]. GI can occur in the form of gross chromoso- mal abnormalities (i.e., chromosomal instability) and/or small-scale genetic changes (e.g., microsatellite instability). As mentioned be- fore (section 4), the majority of Cr-related lung squamous cell car- cinomas analyzed are characterized by microsatellite instability [46]. Rather surprisingly, no microsatellite instability could be de- tected 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 can- cer [172]. This clastogenic action of Cr(VI) compounds is consis- tent 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 impor- tance 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 car- cinogenic response independently of the exposure route [174,179- 186].

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 trans- ported into epithelial cells are those that are slowly and continu- ously released from particulate compounds that adhered to the sur- face 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 epi-

thelium 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 mecha-

nism 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 meta-

plasia in the bronchial epithelium, a transformed state from which

squamous carcinomas may arise [186]. Interestingly, a persistent

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carcinomas may arise [186]. Interestingly, a persistent lung in- flammatory 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 par- ticipate 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 cola- genases [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].

is used [204]. This could also be the basis for the chromate inhibition of pyruvate de- hydrogenase [86], as these two enzymes have this moiety in com- mon [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). Unfor- tunately, with the exception of a preliminary broad study with gly- colytic and pentose phosphate pathway enzymes [132] that revealed no significant differences in activity after Cr(III) and Cr(VI) treat- ment, 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 inhibi- tor 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 sur- veillance and regulatory mechanisms, termed cell cycle check- points, which ensures that, during each cell cycle, DNA replication

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. Elimi- nation 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 under- stood. Reports of intracellular ROS generation upon Cr(VI) expo- sure 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 super- oxide 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 apopto- sis 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 care- fully 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 in- duced by Cr(VI). Deficiencies in these repair systems can be asso- ciated with both the onset and the progression of cancer, as an in- creased frequency of mutations can result in the activation of onco- genes 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 acti- vates base excision repair (BER) and the apurinic/apyrimidinic (AP) endonuclease repair system. BER recognizes damaged (oxi- dized or alkylated) bases and excises them through the use of spe- cific 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 5- deoxyribophosphate [222]. These SSBs are subsequently recog- nized 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 5- deoxyribophosphate 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-

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ingly, exposure of A549 cells to Cr(VI) concentrations (>25 M) highly favorable to ROS generation and, consequently, to 8-oxo-2- deoxyguanosine formation inhibited the expression of OGG1, both at the mRNA and protein levels [144,226]. This might have in- creased 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 me- dium and employing other methodologies to evaluate OGG1 ex- pression and oxidative DNA damage, results could not be repro- duced [76], which may have resulted from differences in the reduc- tive 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 gener-

ated. Overall, these results suggested that: (i) the repair of Cr le- sions by BER is possibly error-prone; (ii) in the absence of a func- tional 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 solu- ble Cr(VI) compounds, it had a strong effect in the case of particu- late compounds [229,230]. The fact that the fagocytic internaliza- tion 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 per- turb 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 replica- tion homologous recombination (HR), base oxidation and methyla- tion 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

[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 mutagene- sis, 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 mismatch- provoked excision reaction directed by a 5 strand break which ter- minates 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 acti- vation and the subsequent excision of up to 1 kb of newly synthe- sized 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 sub- sequent 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 ex- pression, 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 expo- sure to this carcinogen [166,252,265], suggesting a distinct mecha- nism of formation. The fact that XRCC1-deficient (EM9) and pro- ficient (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

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rearrangements and, as a consequence, genomic instability. There- fore, their presence elicits normally a strong apoptotic response [272], which can explain the MMR-dependent mechanism of apop- tosis observed in Cr(VI)-treated cells described by the group of Zhitkovich [252]. Having confirmed that the absence of a func- tional 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 transforma- tion of BEAS-2B cells generated a malignant cell line, RenG2, with a functional MMR system and without MSI [83] is in apparent con- tradiction with this theory. These conflicting results may have re- sulted from: (i) the use of different cell lines (although both of hu- man respiratory tract origin); (ii) the fact that BEAS-2B cells are SV40 immortalized and, as such, have a nonoperational p53 path- way [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 cy- cle. Accordingly, human cells deficient in WRN protein are hyper- sensitive 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 ul- cerations [6]. Considerable data support a role of apoptosis in the remodeling of lung tissue after acute lung injury [276-279]. Inter- estingly, 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 signal- ing pathways that are affected by Cr(VI) downstream of its geno- toxic effects, namely those that regulate cell survival and prolifera- tion, is a key issue to understand the molecular events leading to Cr(VI)-induced carcinogenesis. Gene expression profiles are excel- lent 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 me- tabolism 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 con- ducted 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 expres- sion 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 re-

duced 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

Cr(VI) [282], a decrease in the expression of epidermal growth factor receptor (EGFR) and human epidermal growth factor recep- tor 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 ran-

domly [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 stress- related genes (catalase, glutathione S-transferase, glutathione reduc- tase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxi- dase, 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 activa- tion 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 selec- tive 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 transactiva- tion [293].

14.3.2. Cr(VI) Effects on Transcriptional Initiation

Gene expression is strongly influenced by the activity of tran- scription 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 sev-

eral other transcription factors that activate the transcription of

genes involved in inflammation, carcinogenesis and pro- or anti-

apoptotic pathways were studied both in malignant and non-

malignant 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., cAMP-

response-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].

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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 con- sequences on cell fate due to the opposed roles of these two tran- scription factors in lung disease and innate immune system re- sponse. STAT1 is a downstream effector of interferon (IFN) signal- ing [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 stimu- lates STAT3 and STAT5, providing a survival advantage and lead- ing, possibly, to neoplastic transformation [299,301,302]. Moreo- ver, in the airways, loss of STAT1 is recognized to increase the severity of viral lung infections [303] and metal- or chemical- induced 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 lack- ing 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 fam- ily kinases Lck and Fyn, respectively, and the consequent accumu- lation of the inflammatory cytokine interleukine 6 [293,306], may have important repercursions on lung cancer onset, as chronic in- flammation 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 pref- erentially in nuclear matrix DNA, where many replication, repair and transcription proteins associate, were proposed to play an im- portant role in the epigenetic events associated with Cr(VI) expo- sure [312]. For instance, Cr(VI) cross-linking of histone deacety- lase-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 transactiva- tion [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 trans- activation of CYP1A1 in rat hepatoma cells [292,314] and the in-

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 transac- tivation 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 re- sponse 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 se- quences [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 interfer- ing with the function of all MTF-1 different transactivation do- mains, 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 com- pact chromatin structure of inducible promoters makes them better targets for Cr binding than the more compact chromatin of constitu- tive promoters.

14.3.4. The Involvement of p53 in the Signaling Pathways Acti- vated by Cr(VI)

The tumor suppressor protein p53 is involved in many path- ways that are activated in response to DNA damage, namely cell cycle arrest and apoptosis, and is a key player in neoplastic trans- formation [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, p53- dependent cell cycle arrest could not be observed [218]. A less known mechanism of Cr(VI)-induced apoptosis operates in a p53- independent 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 poten- tial. These events ultimately lead to DNA fragmentation and apop- tosis via an increase of cytosolic Ca

²⁺

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 responsi- ble for the maintenance of proliferative signals and is involved in the early stages of neoplasia [149]. There are over a dozen mam- malian MAP kinase genes. The best known belong to the extracel- lular 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].