Threshold mechanisms and site specificity in chromium(VI) carcinogenesis

Silvio De Flora

Department of Health Sciences, Section of Hygiene and Preventive Medicine, University of Genoa, via A. Pastore 1, I-16132 Genoa, Italy


    Abstract
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Ten years have elapsed since the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of chromium and chromium compounds. Further studies performed during the last decade have provided further epidemiological, experimental and mechanistic data which support the IARC conclusions. A wealth of results indicate that, at variance with chromium(0) and chromium(III), chromium(VI) can induce a variety of genetic and related effects in vitro. The lack of carcinogenicity of chromium(0) and chromium(III) compounds in experimental animals is well established, and only a minority of animal carcinogenicity data with chromium(VI) compounds were positive (30 out of 70, i.e. 42.9%). Moreover, most positive studies used administration routes which do not mimic any human exposure and by-pass physiological defense mechanisms. Typically, positive results were only obtained at implantation sites and at the highest dose tested. Exposure to chromium(VI) has been known for more than a century to be associated with induction of cancer in humans. Carcinogenicity requires massive exposures, as is only encountered in well defined occupational settings, and is site specific, being specifically targeted to the lung and, in some cases, to the sinonasal cavity. Increased death rates for cancers at other sites, which were occasionally reported in some epidemiological studies, were almost invariably not statistically significant, and inconsistent (being counterbalanced by other studies which apparently showed decreased rates for the same cancers). As we recently quantified in human body compartments, chromium(VI) can be reduced in body fluids and non-target cells, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes. In target cells, chromium(VI) tends to be metabolized by a network of mechanisms leading to generation of reduced chromium species and reactive oxygen species, which will result either in activation or in detoxification depending on the site of the intracellular reduction and its proximity to DNA. When introduced by the oral route, chromium(VI) is efficiently detoxified upon reduction by saliva and gastric juice, and sequestration by intestinal bacteria. If some chromium(VI) is absorbed by the intestine, it is massively reduced in the blood of the portal system and then in the liver. These mechanisms explain the lack of genotoxicity, carcinogenicity, and induction of other long-term health effects of chromium (VI) by the oral route. Within the respiratory tract, chromium(VI) is reduced in the epithelial-lining fluid, pulmonary alveolar macrophages, bronchial tree and peripheral lung parenchyma cells. Hence, lung cancer can only be induced when chromium(VI) doses overwhelm these defense mechanisms. The efficient uptake and reduction of chromium(VI) in red blood cells explains its lack of carcinogenicity at a distance from the portal of entry into the body. All experimental and epidemiological data, and the underlying mechanisms, point to the occurrence of thresholds in chromium(VI) carcinogenesis.

Abbreviations: CSC, cigarette smoke condensate; ELF, epithelial-lining fluid; GSH, reduced glutathione; IARC, International Agency for Research on Cancer; PAM, pulmonary alveolar macrophages; RBC, red blood cells; USEPA, US Environmental Protection Agency.


    Introduction
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Ten years have elapsed since the International Agency for Research on Cancer (IARC) evaluated the carcinogenicity of chromium and chromium compounds (1). As I will comment in the present article, nothing new has happened in this decade to change the evaluation made by IARC, which is further supported by recent scientific literature. Recently, the IARC re-evaluated as many as 123 of the 834 agents evaluated in Volumes 1–70, due to the concern that the older evaluations were becoming out of date (2). These 123 compounds were selected either because (i) there was a large quantity of new data and there was specific, widespread interest in having them re-evaluated; (ii) new epidemiological data and/or experimental carcinogenicity data were available, to such an extent that changes in the classification were anticipated; or (iii) new experimental carcinogenicity data had become available which were unlikely to lead to any change in the evaluation and classification (2). Chromium compounds were not included in any of these three lists.


    Potential carcinogenicity of chromium, as inferred from short-term tests and biomarkers
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Short-term tests
A huge literature covers the activity of chromium compounds on genetic and related effects in in vitro test systems. De Flora et al. (3,4) reviewed more than 700 sets of data obtained by testing 32 chromium compounds in as many as 130 experimental systems having distinct end-points and/or cellular targets. The large majority of the results obtained with 17 chromium(VI) compounds (384 out of 436 data, i.e. 88.1%) were positive. The activity was less frequently evident with chromium(VI) compounds having a low solubility, which often needed to be artificially solubilized, e.g. with alkali, prior to challenge with target cells. In contrast, testing of 12 chromium(III) compounds mostly generated negative data. In fact, by excluding those positive results obtained with chromium(III) compounds which had been contaminated with chromium(VI) traces, as recognized by the authors themselves, only 42 out of 203 data (20.6%) were positive. In most studies, the purity of chromium(III) compounds had not been checked, and positive results could often be ascribed either to artifacts or to lack of validation of the test system. In any case, the doses needed for chromium(III) compounds to generate positive data were two or three orders of magnitude higher than those needed for chromium(VI) compounds to yield the same results (1,3). Chromium(0), in the form of chromium carbonyl, is also devoid of mutagenicity in vitro (5).

Therefore, there is overwhelming evidence indicating that, at variance with chromium(III), chromium(VI) compounds are genotoxic and potentially capable of initiating cancer. Unfortunately, short-term tests do not contribute to the elucidation of which chromium(VI) compounds possess these properties in whole organisms, which depends on the availability of chromium(VI) to target cells, as a function of solubility of compounds and of toxicokinetic and metabolic mechanisms.

The differential toxicological relevance of these chromium species mainly depends on the fact that chromium(VI), in the form of chromate anion, which predominates over the dichromate anion at physiological pH, is easily taken up via the general anion channel protein or band 3 protein. In contrast, cellular membranes are normally impermeable to chromium(III) cations, which can penetrate into cells only under particular conditions (6).

Biomarkers of exposure and effect in humans
Chromium levels in the blood and urine are biomarkers of exposure, reflecting the amount of chromium which is internalized in the body. However, whereas chromium in erythrocytes is diagnostic for internal exposure to chromium(VI), it is not possible to distinguish, on the basis of urinary chromium, whether exposure to chromium(VI) or chromium(III) has occurred (7). In fact, as the result of chromium(VI) reduction in the blood, all chromium detectable in the urine of exposed individuals is chromium(III) (1,8,9).

A number of studies evaluated the problem of biological monitoring by assessing cytogenetic end-points, such as chromosomal aberrations, sister chromatid exchanges and micronuclei, in lymphocytes of chromium-exposed workers. In spite of the consistent genotoxicity of chromium(VI) compounds in in vitro test systems, the majority of cytogenetic surveillance studies among chromium platers, ferrochromium workers and stainless steel welders have yielded negative or inconclusive results (reviewed in ref. 7). Similarly, either positive results or negative results were obtained by measuring DNA single-strand breaks in the lymphocytes of chromium-exposed workers (10). No oxidative DNA damage occurred in chromium(VI) production workers (11), and no significant increase of DNA–protein crosslinks was observed in chromium platers (12).


    Evaluation of chromium carcinogenicity in laboratory animals
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Chromium compounds
A number of studies were carried out during the past 45 years in order to evaluate the carcinogenicity of chromium compounds of various solubility and oxidation states in rodents (mainly rats and mice but also hamsters, guinea pigs and rabbits).

Out of the studies reviewed by the IARC (1), 12 results obtained with metallic chromium and 20 results obtained with chromium(III) compounds (chromic acetate, chromic oxide, chromic chloride, chrome tan or basic chromic sulfate, chromic sulfate and chromite) were negative, irrespective of the administration route. Similarly, 27 results available with chromium-containing mixtures and other chromium compounds, one of which relative to chromium(IV) dioxide, were consistently negative. Only one sample of roasted chromite ore, in which chromium(III) was likely to have been oxidized to chromium(VI), was weakly carcinogenic in rats after intrapleural injection (1). Therefore, there is solid documentation that metallic chromium and chromium(III) compounds are devoid of carcinogenicity in laboratory animals.

Several chromium(VI) compounds were assayed for carcinogenicity. Only a minority of the available results (30 out of 70, i.e. 42.9%) were positive (1), which is a rather unexpected outcome if one takes into account the consistent genotoxicity of chromium(VI) when challenged in vitro with target cells. Besides the low frequency of positive results, other patterns are evident. The first is that there is a cluster of positive results corresponding to chromium(VI) compounds of intermediate solubility, such as calcium chromate, strontium chromate and zinc yellow (basic zinc chromate). Another feature is that the majority of positive results were generated in studies in which chromium(VI) compounds were given by certain administration routes (subcutaneous, intramuscular, intraperitoneal, intrapleural, intrabronchial) which not only do not reproduce any human exposure but also by-pass important detoxification mechanisms of the organsim. In addition, all positive results were rather weak and were generated at single, high doses. When more than one dose was tested, no dose–response curve could be drawn. In addition, chromium(VI) only induced local tumors at the administration site, and not at a distance from the portal of entry into the organism. The only exception was a study (13) in which intramuscular injections of lead chromate in rats resulted in the development of renal carcinomas. However, as commented by the IARC Working Group (1), this effect should be ascribed to the lead moiety, which typically produces kidney tumors in rodents (14).

All these patterns are consistent with the occurrence of threshold mechanisms in chromium(VI) carcinogenesis.

Chromium-contaminated environmental samples
Besides several results obtained with mixed chromium dust, all of them negative (1), a recent study (15) specifically addressed the problem of evaluating the carcinogenicity in rats of a soil, which was heavily contaminated with chromium(VI) (almost 6 g/kg soil). The soil sample was collected from Hudson County, NJ, which, during much of this century, was a major center for the processing of chromium ore. After repeated intratracheal instillations, no tumor was detected in rats treated with either control soil or Hudson County soil, the latter receiving a total of 324 µg chromium(VI)/kg body wt. In the same study, intratracheal instillations of calcium chromate, for a cumulative dose of 8.7 mg/kg, and of Hudson County soil supplemented with calcium chromate, for a total of 8.0 mg/kg, produced four and one lung tumors, respectively. These data were not statistically significant (15).


    Carcinogenicity of chromium to humans
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Categorization of chromium compounds according to carcinogenicity
According to the World Health Organization (WHO), `chromium(III) is not considered to be carcinogenic' (16). The IARC evaluated that `there is inadequate evidence in humans for the carcinogenicity' of chromium(0) and chromium(III) compounds. Also based on the results of animal carcinogenicity studies, the overall evaluation was that chromium(0) and chromium(III) `are not classifiable as to their carcinogenicity to humans (Group 3)' (1).

In contrast, chromium(VI) has been known to be an occupational carcinogen for more than a century (17). The IARC evaluated that `there is sufficient evidence in humans for the carcinogenicity of chromium(VI) compounds', as encountered in specific occupational settings. The overall evaluation was that `chromium(VI) is carcinogenic to humans (Group 1)', as inferred from `the basis of the combined results of epidemiological studies, carcinogenicity studies in experimental animals, and several types of other relevant data which support the underlying concept that chromium(VI) ions generated at critical sites in target cells are responsible for the carcinogenic action observed' (1). The US Environmental Protection Agency (USEPA) has also classified chromium(VI) in Group A, that is a human carcinogen (18).

Workers at risk
A number of occupational activities involve the production or use of chromium or chromium compounds (1). An estimated 305 000 workers in the USA are potentially exposed to chromium and chromium-containing compounds in the workplace (19).

The epidemiological evidence for an increased risk of respiratory cancer in chromium(VI)-exposed workers was provided under conditions of high exposure, as encountered in the chromate production, chromate pigment production and chromium plating industries, which in the past decades mainly involved mixed exposures to various chromium(VI) compounds and possibly to other metals as well as organic substances. In contrast, no evidence is available for carcinogenicity of chromium in other occupational categories (1,19,20). The epidemiological studies do not clearly implicate specific compounds, but do implicate chromium(VI) compounds (19).

Environmental exposures
There is no evidence that chromium causes cancer in humans following environmental exposures. According to WHO, `there is not reason, at present, to be concerned that chromium in the air presents a health problem, except under conditions of industrial exposure' (16).

A retrospective environmental epidemiological study in Sweden `could not demonstrate an increase in lung cancer mortality among a population exposed to exhaust from ferrochromium industries' (21). Two studies raised the suspicion that contamination of water with chromium(VI) could be associated with an increased mortality for cancer, but in both cases the same authors ruled out this hypothesis. Thus, after a preliminary study reporting increased incidences of cancer in a population who resided in a polluted area near an alloy plant that smelted chromium in the People's Republic of China (22), no chromium-related increases in stomach cancers or overall cancer mortality could be documented in a more accurate evaluation (23). Between 1966 and 1986, the childhood leukemia rate in Woburn, MA, was higher than the national average (24). This finding prompted a multidisciplinary research team from MIT, which was supported by the NIEHS Superfund Basic Research Program. Measurement of arsenic and chromium in hair samples from Woburn residents ruled out any role of contamination with these metals of drinking water from a municipal supply (25).

Target organs
As specified previously, the epidemiological studies carried out in certain occupational settings consistently show that (unidentified) chromium(VI) compounds can be carcinogenic for the lower respiratory tract. Moreover, a few studies reported an association between exposure to chromium(VI) and cancers of the sinonasal cavity. An increased mortality for cancers at other sites was sporadically reported, which was counterbalanced by several studies in which the risk of developing cancer at various sites was apparently decreased in chromium(VI)-exposed individuals. Clearly, these findings reflect the lack of consistency of some data generated in epidemiological studies performed in relatively small groups of exposed workers. Moreover, most occupations involved exposures not only to chromium(VI) compounds but also to other recognized carcinogens, such as other metals (e.g. nickel), organic compounds (e.g. polycyclic aromatic hydrocarbons), fibers (e.g. asbestos) and complex mixtures (e.g. cigarette smoke). It is thus important to discriminate the effects of confounding factors and to pinpoint those findings that are validated by the converging evidence provided by different epidemiological studies.

Based on these considerations, in 1988 the WHO concluded that `there is insufficient evidence to implicate chromium as a causative agent of cancer in any organ other than the lung' (16). In 1990, the IARC concluded that `for cancers other than of the lung and sinonasal cavity, no consistent pattern of cancer risk has been shown among workers exposed to chromium compounds' (1). This conclusion was reiterated, almost verbatim, in a review article published 3 years later (26). A recent authoritative article on carcinogenicity of metals in humans (20) further emphasized the selective carcinogenicity of chromium(VI) in the lung and nasal cavity of workers in chromate production, chromate pigment production and chrome plating. In the same year, however, another review article claimed that chromium(VI) can be involved in the causation of a broad spectrum of additional cancers, including prostate cancer, lymphoma, leukemia, and bone, stomach, brain, kidney and testicle cancers (27). This position is surprising not only for the lack of scientific support, as discussed below, but also because the same author had been a member of the IARC Working Group (1) and had 4 years earlier co-authored the article (26) supporting the selective carcinogenicity of chromium(VI) to the human lung and sinonasal cavity.

Eleven epidemiological studies were cited by Costa (27) to support the broad target carcinogenicity of chromium(VI) in humans. Four of these studies (2831) had previously been evaluated by the IARC (1), whereas the remaining seven studies (24,3237) are more recent. The possible exposures were extremely heterogeneous, and in most instances they also included agents other than chromium. Ten studies (2837) covered seven different occupational settings, whereas one study referred to a possible exposure to groundwater contaminated with arsenic and chromium (24). It is noteworthy that the responsibility of these metals for the increased mortality of leukemia in children and youngsters had been presented as a working hypothesis (24), which was later demonstrated to be unjustified by the same group (25).

The Costa (27) paper suffers from a number of inaccuracies and gross mistakes, regarding references, working categories, identity or definition of cancer sites and report of data. Moreover, Costa (27) arbitrarily selected those data which could be of some use to support his theories, i.e. just two data for prostate cancer, one for cancer of the haemolymphopoietic system, one for Hodgkin's disease, three for leukemia, one for bone cancer, five for stomach cancer, four for brain cancer, one for bladder cancer, two for renal cancer and two for genital cancer. In contrast, he disregarded other data reported in the same papers, many of which showed negative or even opposite trends. For instance, in one study only (30) was there a significant increase of stomach cancer deaths, and in white workers only, while in another study (36) the observed stomach cancer deaths were significantly lower than expected. Therefore, in spite of exposures to chromium(VI) compounds of workers by the oral route, due either to impact of large size particles onto the oropharyngeal mucosa or to swallowing of particles refluxed from the respiratory tract via the muco-ciliatory escalator, recent data are consistent with the consensus reached by the IARC Working Group (1) that there is no consistent indication in the literature supporting an increased risk of stomach cancer.

Very few of the increases in mortality rates claimed by Costa (27) are statistically significant, and in most cases they appear to be biologically irrelevant and due to chance. Occasional decreases of the mortality rates or even, in many cases, the unexpected lack of deaths for cancers at given sites should be interpreted in the same way. No cancer in the sinonasal region was reported in any of these studies, and even cancers in the lower respiratory tract were significantly increased in a limited number of studies, including one only of two studies in chrome platers (29), one only of two studies in workers exposed to lead and zinc chromate pigments (32) and one study in zinc chromate painters (31). On the other hand, no significant excess risk was observed in two studies in ferrochromium workers (33,34), one study in welders (35) and one study in tanners (36). On the whole, these recent data fully support the selective respiratory carcinogenicity of chromium(VI) compounds in the three aforementioned categories of chromium(VI)-exposed workers (1,19,20).


    Mechanistic interpretations based on chromium(VI) toxicokinetics and metabolism
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Reduction of chromium(VI) in body fluids and non-target cells
The chromium(VI) reducing capacity of rat liver preparations was shown independently >20 years ago by Gruber and Jennette Wetterhahn (38) and in my laboratory (39,40) using analytical tecniques and mutagenicity assays, respectively. At the same time, we demonstrated that it is not possible to oxidize inactive chromium(III) to mutagenic chromium(VI) by means of a variety of rat and human metabolic systems (41).

After that, we evaluated the chromium(VI) reducing capacity of metabolic systems derived from humans and a variety of other animal species, including rodents (mice, rats, hamsters, woodchucks), avian species (chicken, Pekin duck) (reviewed in ref. 6), freshwater fish (42), seawater fish (43) and even marine sponges (44). Using either analytical techniques and/or mutagenicity assays, we evaluated the fate of chromium(VI) after challenge with body fluids, such as saliva, gastric juice and epithelial-lining fluid (ELF); microorganisms colonizing the human body, such as intestinal bacteria; isolated cells, such as red blood cells (RBC) and bronchoalveolar lavage cells, mainly consisting of pulmonary alveolar macrophages (PAM); tissues and organs, such as liver, peripheral lung parenchyma, bronchial tree, cutis, subcutis, kidney, testis, stomach, intestine, spleen, bladder and adrenals (5,6,45).

The chromium(VI) reducing capacity, as inferred from ex vivo studies, was expressed in quantitative terms in each study. Based on these data, we recently made quantitative estimates of the overall reducing capacity of chromium(VI) in some human body compartments by relating the specific reducing activity of body fluids, cell populations or organs to their average volume, number or weight (45). Reduction of chromium(VI) in body fluids and non-target cells is expected to greatly attenuate or eliminate its potential toxicity and carcinogenicity, to imprint a threshold character to the carcinogenesis process, and to restrict the possible targets of its activity.

Metabolism of chromium(VI) in target cells
The overall evaluation of the IARC Working Group on chromium(VI) compounds underlies the `concept that chromium(VI) ions generated at critical sites in the target cells are responsible for the carcinogenic action observed' (1). This concept was reiterated verbatim by the US Department of Health and Human Services (19). Which are target cells? They are those cells which can be exposed to chromium(VI) escaping upstream reduction in body fluids and non-target cells. Therefore, as previously discussed, any prokaryotic or eukaryotic cell can be the target for chromium(VI) genotoxicity in vitro; in animal carcinogenicity assays, target cells are those where chromium(VI) is locally applied; in humans exposed to high doses of chromium(VI), target cells are those of the respiratory tract, provided chromium(VI) is successful in overwhelming the defense mechanisms of airways. Thus, the selection between target cells and non-target cells is operated by toxicokinetic factors affecting reduction of chromium(VI).

Once in touch with cells, the chromate anion easily crosses cell membranes (6), after which chromium(VI) tends to be reduced inside the cell. Thus, chromium(VI) functions as a sort of Trojan horse, allowing penetration of chromium into the cell. Other types of cellular uptake, such as internalization of insoluble particles, have also been shown to occur in vitro (46). Chromium(VI) is reduced in different cell compartments. According to our studies, reduction in the cell cytosol is greater than in the endoplasmic reticulum and in mitochondria and the nucleus (47). Chromium(VI) reduction is a composite process due to a network of mechanisms, which involve the contribution of reducing molecules, such as ascorbate, glutathione (GSH), cysteine, hydrogen peroxide and riboflavine, as well as enzyme-catalyzed reactions, e.g. by cytochrome P450, aldehyde oxidase and DT diaphorase (reviewed in ref. 6).

Reduction of chromium(VI) results in the formation of chromium(III), the stable reduced form, which binds DNA more efficiently than chromium(VI) (3). Moreover, intermediate reduced products, i.e. chromium(V) and chromium(IV), are also suspected to play a role in chromium genotoxicity and carcinogenicity, either per se or through reaction with other cellular components resulting in the generation of reactive oxygen species, and in particular of the hydroxyl radical (OH·) (reviewed in ref. 6). Oxidative DNA damage is detectable after exposure of cells to chromium(VI) in vitro (6,46,48,49), and we have recently shown its occurrence in vivo in lung cells but not in liver cells of rats receiving i.t. instillations of chromium(VI) (50). Unfortunately, many studies using acellular systems, in which chromium(VI) and a reducing system are mixed together in a tube, in close contact with pure DNA, do not take into account the extreme reactivity of OH·, which does not live >1 ns and does not travel >1 nm (51). Therefore, this mechanism is likely to work in whole cells only when OH· generation occurs in very close contact with DNA. Several types of DNA damage occur in chromium(VI)-exposed cells, including single-strand breaks, DNA–DNA interstrand crosslinks, DNA–protein crosslinks, chromium–DNA adducts, oxidative nucleotide changes and chromosomal aberrations (6,46).

Chromium(VI) reduction in target cells may be variously interpreted. When Gruber and Jennette Wetterhahn (38) showed that chromium(VI) can be reduced by rat liver P450 enzymes, they raised the hypothesis that this is an activating mechanism. This theory (reduction–activation) is quite plausible since, as just discussed, the reduced species are the most likely DNA-damaging agents. In the same year, I demonstrated that rat liver preparations decrease chromium(VI) mutagenicity (39), and I interpreted this finding as a detoxification mechanism. This theory (reduction–detoxification) is also supported by several lines of evidence, such as the fact that tissues or organs, such as the liver, which are not the target for chromium(VI) carcinogenicity, are characterized by a potent chromium(VI) reducing capacity in a broad spectrum of animal species. In contrast, typical targets for chromium(VI) carcinogenicity in rodent assays, such as the rat skeletal muscle, have a negligible effect on chromium(VI) mutagenicity (40). Therefore, tissue-specific susceptibility to chromium(VI) carcinogenicity correlates with intrinsically poor capacity for intracellular chromium(VI) reduction. Probably, both theories are valid. When in 1989 Karen Wetterhahn and myself prepared an extensive review article covering the mechanisms of chromium metabolism and genotoxicity, we tried to harmonize our positions (see Chapter 7.3 and Figure 54 of ref. 6). We reached a consensus that the intracellular chromium(VI) reduction, leading to generation of reactive species, may be viewed as an activation process, when it occurs in the proximity of DNA. Alternatively, reduction is a detoxification process when it occurs far away from DNA, and the reactive species can be trapped by a large number of ligands, nucleophiles and antioxidants which are present in the intracellular environment. Therefore, the cellular site of reduction is crucial in affecting the fate of the cell taking-up chromium(VI) (6). It is also noteworthy that chromium(VI)-exposed cells can undergo apoptosis (46) as a consequence of DNA damage, and therefore are eliminated from the organism.


    Fate of chromium(VI) in the organism
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 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
Exposure by the oral route
Chromium(VI) can be ingested with drinking water, other beverages and food. Certainly, many food components have reducing properties. It has been shown that chromium(VI) is efficiently reduced when drinking water is used to prepare common beverages (52). Another way of exposure of the digestive tract is swallowing of chromium(VI) refluxed from airways via the muco-ciliatory escalator in individuals exposed by inhalation.

Ex vivo studies performed in my laboratory documented the chromium(VI)-reducing capacity of the human digestive tract (45). In particular, we estimated that saliva reduces 0.7–2.1 mg chromium(VI)/individual/day and gastric juice reduces at least 80.3–84.5 mg chromium(VI)/individual, as assessed by evaluating the circadian course of chromium(VI) reduction in volunteers (53). Reduction of chromium(VI) by gastric juice is due to thermostable components of gastric secretions, and is favored by low pH. The reaction is complete within 10–20 min, and at least half of it is accomplished in 1 min (45). We also estimated that the daily removal of chromium(VI) with fecal bacteria is 15.4–33.4 mg/individual (45). Intestinal bacteria contain high amounts of GSH (54), which is an efficient reductant for chromium(VI) (6). Since bacterial GSH is released extracellularly (54), it is likely that chromium(VI) is additionally reduced in the intestinal lumen.

Reduction of chromium(VI) in the digestive tract explains why, in both humans and animal models, there is a very poor intestinal absorption of orally introduced chromium(VI) (8,5557). In case chromium(VI) may escape reduction by saliva and gastric juice, sequestration by intestinal bacteria and, probably, other mechanisms that we did not investigate, it will be absorbed by the intestine, released into the blood of the portal system, and carried to the liver. As described below, the blood, and in particular RBC, has a considerable capacity of sequestering and reducing chromium(VI). The human liver can reduce as much as 3.3 g chromium(VI)/individual (45). Although it is impossible to estimate the daily reduction, it should be kept in mind that many of the chemical and biochemical mechanisms accounting for chromium(VI) reduction in the liver are continuously renewed.

The acute oral toxicity of chromium(VI) is rather low, the LD50 in rodents being >50 mg chromium(VI)/kg body wt (58). Few lethal effects were observed in episodes of accidental ingestion or tentatives of suicides with high doses of chromium(VI). For instance, following ingestion of chromium(VI) compounds in the 8–20 g range, eight subjects survived and three subjects died (59). Oral chromium(VI) is not genotoxic at doses which greatly exceed the drinking water standards. Thus, ingestion of a bolus dose of 5 mg chromium(VI) by human volunteers did not result in any increase of DNA–protein crosslinks in peripheral blood lymphocytes (60). Administration of up to 20 mg chromium(VI), either in drinking water or by gavage, failed to produce any effect in the mouse bone marrow micronucleus assay or in the rat hepatocyte DNA repair assay (61). As discussed previously, the lack of carcinogenicity of chromium by the oral route is supported both by animal studies and epidemiological data. As to other health effects resulting from long-term exposures to chromium(VI) with drinking water, no adverse effect was observed in dogs fed 11.2 mg/l chromium(VI) for 4 years (62) or rats fed 25 mg/l for 1 year (63) or 134 mg/l for 6 months (64). Moreover, no health effect was reported in a family accidentally exposed to chromium(VI) at 1 mg/l in well water for 3 years (65).

Exposure by inhalation
The respiratory tract is the only target for chromium(VI) carcinogenesis in individuals performing certain occupational activities, which involve exposures to levels in air that were several orders of magnitude higher than those found in the natural environment (1,19). This conclusion, together with the inconsistent positive results obtained in rodents exposed to chromium(VI) by inhalation or intratracheal instillation, suggests that respiratory cancer can only be induced by chromium(VI) doses which overwhelm the body's defense mechanisms. It is well known that the respiratory tract has important non-specific defense mechanisms (66). In addition, we demonstrated that chromium(VI) reducing mechanisms occur in the lower respiratory tract. In particular, the ELF, including the surfactant and bronchial–bronchiolar secretions which can be recovered by bronchoalveolar lavage, has an overall reducing capacity of 0.9–1.8 mg chromium(VI) (45,67). The ELF of both humans (68) and rats (69) is known to be particularly rich in antioxidants.

PAM are sweeping cells which can reduce ~2 µg chromium(VI)/106 cells in rats (70) and 4.4 µg chromium(VI)/106 cells in humans (67). Based on the knowledge that 23x109 PAM populate terminal airways in humans, we estimated an overall chromium(VI) reducing capacity of 136 mg per individual (45). Since 1—5x106 PAM are removed every hour from human terminal airways via the muco-ciliatory escalator (66), it can be calculated that the PAM population which is renewed every day will reduce 0.1—0.5 µg chromium(VI). Thus, even in cases of long-term exposure, which require a continuous detoxification of inhaled chromium(VI), the respiratory tract appears to have efficient reducing mechanisms, which, however, are not as formidable as those of the digestive tract.

Human bronchial tree (71) and peripheral lung parenchyma (72) showed a notable chromium(VI) reducing activity. Since these tissues are possible targets for chromium(VI) carcinogenicity, the outcome of the chromium(VI) reduction, either activation or detoxification, will depend on the intracellular site where the process is accomplished. Since peripheral lung parenchyma reduces on average 0.2 mg chromium(VI)/g (72), and the human lungs weigh 1300 g, we estimated an overall chromium(VI) reducing capacity of 260 mg per individual (45).

Auto-induction of chromium(VI) metabolism in the lung
An interesting issue is the possible modulation of the pulmonary metabolism of chromium(VI) and occurrence of interactions with other inhalable carcinogens. We demonstrated that repeated intratracheal instillations of sodium dichromate in rats, according to a schedule which failed to induce lung tumors (73), result in an auto-induction of chromium(VI) metabolism in the lung (74). In other words, repeated exposures to chromium(VI) potentiate its own metabolism in the lung (74), and this correlates with failure to induce lung tumors under the same experimental conditions (73). Incidentally, this is a further finding which supports the role of chromium(VI) reduction in lung cells as a detoxification mechanism.

Interaction between chromium(VI) and cigarette smoke
The interaction between airborne exposure to chromium(VI) and cigarette smoke is an intriguing problem, which was not clarified by epidemiological studies (17). No synergistic effect between cigarette smoking and stainless welding was observed in the induction of chromosome damage in lymphocytes (75). Smokers usually show higher urinary chromium levels than non-smokers (7), which might be ascribed to enhanced retention of particulates in the bronchial tree. Alternatively, this finding may be interpreted as a stimulation of chromium(VI) reduction in smokers, leading to an increased urinary excretion of chromium(III). In fact, we showed that a cigarette smoke condensate (CSC) decreased the mutagenicity of chromium(VI) in bacteria, presumably due to the presence of reducing agents in CSC. At the same time, chromium(VI) inhibited the metabolic activation of either CSC or benzo[a]pyrene to mutagenic derivatives (5). The antagonistic effect between chromium(VI) and benzo[a]pyrene diol epoxide was recently shown by also evaluating the frequency of HPRT mutants in cultured human fibroblasts (76).

Moreover, the PAM from current smokers exhibited a chromium(VI) reducing capacity which was significantly higher than that of either ex-smokers or never smokers (67). Similarly, preparations of peripheral lung parenchyma from smokers were significantly more efficient than those from non-smokers in decreasing chromium(VI) mutagenicity (72). Therefore, there are some lines of evidence suggesting that, at least in certain steps of the carcinogenesis process, there is a less than additive effect between chromium(VI) and cigarette smoke. Recently, using an acellular system, i.e. by reacting a CSC, chromium(VI) and plasmid DNA, it was shown that this mixture results in an enhanced generation of OH· radicals and induction of DNA single-strand breaks (77). However, as discussed previously, this type of study suffers from the limitation that OH· generation is artificially achieved in close contact with DNA rather than in a whole organism. We have now in progress a study in rats exposed whole-body to cigarette smoke and/or treated intratracheally with chromium (VI). We will evaluate several biomarkers, including oxidative DNA damage, DNA–protein crosslinks, smoke-related and chromium(VI)-related DNA adducts in lung cells, and cytogenetic damage in PAM and bone marrow cells. The results of cytogenetic analyses clearly showed that the combined exposure to cigarette smoke and chromium(VI) is less than additive in enhancing the frequency of micronuclei in PAM and bone marrow cells (R. Balansky, F. D'Agostini, A. Izzotti and S. De Flora, manuscript in preparation).

Dermal exposure
Together with inhalation and oral penetration, dermal contact is the only possible route of exposure of humans to chromium(VI). It is well known that direct skin contact with chromium(VI) may produce irritating and ulcerating effects and elicit an allergic response, characterized by eczema and dermatitis, in sensitized individuals (19). No studies were located regarding cancer in humans or animals after exposure to chromium compounds (19). However, it is very likely that at least a proportion of workers involved in chromium production and use were exposed not only via the respiratory route but also by the oral route and by the dermal route as well. In this connection, it is noteworthy that no significant increase in mortality for skin cancer (35) or specifically for malignant melanoma (33) could be detected in chromium(VI)-exposed workers.

Lack of carcinogenicity of chromium(VI) at a distance from the penetration site
As previously commented, it is typical that, even when chromium(VI) was successful in inducing tumors in experimental animals, they only occurred at implantation sites. In humans, the respiratory tract is the only target for chromium(VI) carcinogenicity which was consistently documented by epidemiological studies.

The explanation for the lack of carcinogenicity of chromium(VI) at a distance from the penetration site is that, in case chromium(VI) may escape detoxification in the respiratory tract, it will be released into the blood stream. Therefore, chromium can be transported to any organ via the blood circulation. In particular, while chromium(III) is transported in the blood plasma, bound to proteins, such as transferrin (6), chromium(VI) is selectively accumulated in RBC. This is so typical that, for half a century, radioactive chromium (51Cr) has been used to tag RBC (78). After penetration into RBC, chromium(VI) is reduced to chromium(III), especially by GSH, and bound to low molecular weight components and chiefly to hemoglobin (6,8,52,57,7983).

In 1978 we demonstrated that chromium(VI) mutagenicity is lost in the presence of human RBC lysates (40). Recently, we evaluated that whole blood has an overall sequestering capacity of 234 and 187 mg chromium(VI)/individual, and that RBC reduce at least 138 and 100 mg chromium(VI)/individual in males and females, respectively. The lifespan (mean ± SD) of human RBC is 107 ± 12 days (84). Therefore, every day a population of 19 ml in males and 14 ml in females will be renewed, thus accounting for at least 1.3 and 0.9 mg chromium(VI) reduced just by the proportion of RBC which is renewed daily. Also in `old' RBC, however, there is a continuous replenishment of GSH, which is imported from the liver (85).

It is thus extremely unlikely that chromium(VI) can travel at a distance from the portal of entry into the organism without having been reduced to chromium(III) upstream in the blood. In fact, both in occupationally exposed workers (9) and in human volunteers drinking chromium(VI)-contaminated water (8), all chromium detectable in urine was chromium(III). In experimental animals `excretion occurs rapidly, and primarily via the kidneys, once chromium(VI) is reduced. Thus, absorption, distribution, metabolism, and excretion of chromium is fairly well understood' (19).


    Multiple lines of evidence supporting the occurrence of threshold mechanisms in chromium(VI) carcinogenesis
 Top
 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 
The issue of thresholds in carcinogenesis, especially in the case of genotoxic carcinogens, is quite controversial. In the case of chromium(VI), even disregarding possible mechanisms occurring after occurrence of DNA damage, e.g. at the level of DNA repair, apoptosis, cell replication or promotion, there seems to be no doubt that toxicokinetic patterns, which restrict the availability of chromium(VI) to certain tissues, and metabolic patterns, which affect the availability of chromium(VI) to DNA, imprint a threshold character to the carcinogenesis process.

The mechanisms described in this article are widely acknowledged in the international literature, and are recognized to represent formidable barriers against chromium(VI) toxicity, genotoxicity and carcinogenicity (52,8689). The IARC Working Group `interpreted these findings as indicating mechanisms that limit the activity of chromium(VI) compounds in vivo' (1). The US Department of Health and Human Services indicated that these `mechanisms limit the bioavailability and attenuate the potential effects of chromium(VI) compounds in vivo. Thus, the oral toxicity of chromium is low' (19). Although 15 years ago the USEPA suggested a dose–response model that is linear at low doses for respiratory cancer (90), this conservative, low dose linearized multi-stage model is out of date and contrary to any mechanistic, epidemiological and experimental evidence in the case of chromium(VI) carcinogenesis. USEPA tends at present to develop more reliable models which take into account mechanistic issues, including toxicokinetics and metabolism (91). In fact, when setting up new standards for drinking water, the USEPA took into account the results of our studies, and concluded that `the body's normal physiology provides detoxification for chromium(VI)' (92).

Even within in vitro test systems, chromium(VI) compounds were often less mutagenic in mammalian assays than in bacterial assays (26,88), which is likely to reflect the greater metabolic capacity of eukaryotic cells as compared with prokaryotic cells, resulting in more efficient detoxification. Likewise, metabolic patterns can explain the lower DNA damage detected in a whole organism than in cultured cells, as demonstrated even when analysing the same end-point in the same type of cells (93). In whole organisms, the potential carcinogenicity of chromium(VI) compounds can be circumvented by the body's defense mechanisms. In humans, this results in a specific localization of cancer to the respiratory tract, and only in those individuals who are exposed to massive doses of chromium(VI) in certain occupational settings. In animal models, the existence of threshold mechanisms is strongly supported by the lack of carcinogenicity of chromium(VI) compounds in the majority of the studies available in the literature. In positive studies, induction of cancer typically occurred under particular conditions overwhelming the defense mechanisms, i.e. (i) at extremely high doses, yielding a yes or no response, without any graduation of the effect; (ii) at implant sites only, and not at a distance from the portal of entry into the organism; (iii) in certain targets only, selected by the potency of detoxification mechanisms; or (iv) when given in a single massive dose, and not in the same cumulative amount fractionated into smaller doses which can be more easily detoxified. These are exactly the patterns that, 15 years ago, I proposed as general criteria for assessing the existence of thresholds in the case of genotoxic compounds which tend to be detoxified in the organism (94).


    Acknowledgments
 
I thank Dr M. Bagnasco for her skillful assistance in preparation of this article. Preparation of this article was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).


    Notes
 
Email: sdf{at}unige.it


    References
 Top
 Abstract
 Introduction
 Potential carcinogenicity of...
 Evaluation of chromium...
 Carcinogenicity of chromium to...
 Mechanistic interpretations...
 Fate of chromium(VI) in...
 Multiple lines of evidence...
 References
 

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Received September 16, 1999; revised December 10, 1999; accepted December 17, 1999.