A tiered approach to investigate the inhalation toxicity of cobalt substances. Introduction: Cobalt's essential role in nature and technology.

Cobalt occurs naturally in the earth's crust, is essential to some microorganisms and forms the core of vitamin B12. Cobalt substances are used in numerous technologies, such as catalysts or batteries. Some of these substances are classified as Carcinogens, while other cobalt compounds have a hazard profile that is less understood and are missing long term studies like cancer bioassays. There is a strong interest by society and industry to reduce and -where possible- eliminate animal testing, yet a necessity by industry and authorities to have sufficient data for hazard conclusions and risk assessments. The present paper introduces a strategy for a mode of action-informed tiered testing, aimed at full inclusion of existing hazard data and selection of relevant biological events towards a certain adverse outcome, i.e. inhalation carcinogenicity. The occurrence of these events following exposure to various cobalt substances is investigated with in vitro and with limited in vivo testing. The tiers of testing are described in the companion papers of this RTP special issue. This approach has given rise to the formulation of two distinct groups, containing substances with similar properties, that can be addressed with limited higher tier animal testing and read across of in vivo results.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 4: Effects from a 28-day inhalation toxicity study with tricobalt tetraoxide in rats.

Lung cancer following inhalation in rodents is a major concern regarding exposure to cobalt substances. However, little information is available on adverse effects and toxicity following long-term inhalation exposure to poorly soluble cobalt substances with low bioavailability. Thus, the present study focused on pulmonary effects of the poorly soluble tricobalt tetraoxide (5, 20, 80 mg/m³) in a 28-day inhalation exposure study. Lung weights increased with increasing exposures. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation at the mid-dose with increasing severity in the high-dose group and post-exposure persistency. Markers for cellular damage and cell proliferation were statistically significantly increased. No increase in 8-OH-dG lesions was observed, indicating an absence of oxidative DNA lesions. The primary effect of inhaled Co3O4 particles is inflammation of the respiratory tract strongly resembling responses of inhaled "inert dust" substances, with a NOAEC of 5 mg/m³ under the conditions of this test.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 3: Inflammatory response following acute inhalation exposure correlates with lower tier data.

In vitro studies have shown that cobalt substances predominantly induce pre-inflammatory biomarkers, resulting in a grouping of substances either predicted to cause inflammation following inhalation, or those with a different reactivity profile (poorly-reactive). There is a lack of data on whole-organ lung responses following inhalation of these substances, especially relating to the poorly-reactive group. It is of interest to generate tissue-specific histopathological correlation to better ascertain the predictive nature of the lower tier tests (i.e. tier 1 - bioelution, tiers 2a and b - in vitro markers and ToxTracker testing), in order to understand the type of effects caused by the poorly-reactive group and to gauge long-term effects. Eight cobalt substances were tested in vivo in a customized 4-h toxicity test; with animals sacrificed up to 14-days post-exposure. Histopathological severity scores were assigned based on inflammatory and pre-carcinogenic markers. A clear pattern emerged, with the reactive substances causing a persistent increase in one or more of the selected markers, and absence of these markers with poorly-reactive substances. Longer-term studies should be conducted to investigate the repeated dose effects of the poorly-reactive substances.

The underlying mode of action for lung tumors in a tiered approach to the assessment of inhaled cobalt compounds.

A mode of action (MOA) for cobalt substances based on the "International Programme on Chemical Safety Conceptual Framework for Evaluating a MOA for Chemical Carcinogenesis" is presented. The data recorded therein were generated in a tiered testing program described in the preceding papers of this special issue, as well as data from the public domain. The following parameters were included in the evaluation: solubility of cobalt substances in artificial lung fluids (bioelution), in vitro biomarkers for cytotoxicity, reactive oxygen species and hypoxia mimicry, inhalation toxicity following acute exposure and repeated dose inhalation effects. Two distinct groups of cobalt substances emerged: substances inducing all effects across the MOA form one group, associated with the adverse outcome of lung cancer in rodents upon chronic exposure. Another group of cobalt substances induces no or very limited effects in the in vitro and acute testing. Higher tier testing with a representative of this group, tricobalt tetraoxide, showed a response resembling rat lung overload following exposure to high concentrations of poorly soluble particles. Based on the fundamental differences in the lower tier toxicological profile, cobalt substances with an unknown hazard profile can be assigned to either group based on lower tier testing alone.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 1: Bioaccessibility testing.

Bioelution tests measure in vitro the release of metal ion in surrogate physiological conditions (termed "bioaccessibility") and estimate the potential bioavailability relative to that of a known reference metal substance. Bioaccessibility of cobalt ion from twelve cobalt substances was tested in three artificial lung fluids (interstitial, alveolar and lysosomal) to gather information about the substances' fate and potential bioavailability in the respiratory tract after inhalation. The results can be used as one line of evidence to support grouping and read-across for substances lacking in vivo data, and where in vivo testing is not readily justifiable. Strong differences were observed in the dissolution behaviour of the substances in the different fluids, with the cobalt substances generally being less soluble in neutral pH fluids and more soluble in the acidic pH fluid. The resulting database, presented with its strengths and limitations, was used to support the formulation of an initial grouping of these cobalt substances into three categories.

Bioaccessibility of nickel and cobalt in synthetic gastric and lung fluids and its potential use in alloy classification.

This study investigated nickel and cobalt ion release from the metals and several alloys in synthetic gastric, as well as interstitial and lysosomal lung fluids. Results were used to calculate the relative bioaccessible concentrations (RBCs) of the metals. Nickel release from SS 316L powder in gastric fluid was >300-fold lower than from a simple mixture of powders of the same bulk composition. Gastric bioaccessibility data showed 50-fold higher metal releases per gram of sample from powder than massive forms. RBCs of nickel and cobalt in the alloy powders were lower, equal, or higher in all fluids tested than their bulk concentrations. This illustrates the fact that matrix effects can increase or decrease the metal ion release, depending on the metal ingredients, alloy type, and fluid, consistent with research by others. Acute inhalation toxicity studies with cobalt-containing alloy powders showed that the RBC of cobalt in interstitial lung fluid predicted acute toxicity better than bulk concentration. This example indicates that the RBC of a metal in an alloy may estimate the concentration of bioavailable metals better than the bulk concentration, and the approach may provide a means to refine the classification of alloys for several human health endpoints.

Copper alloys' metal migration and bioaccessibility in saliva and gastric fluid.

The oral bioaccessibility of copper alloys and pure metals was assessed using in vitro methods with synthetic saliva and gastric fluid. The metal-specific migration rates from polished alloy surfaces are higher in gastric (pH 1.5) than in saliva fluid (pH 7.2). In both media, migrations are higher for lead than for other metals. The bioaccessible metal concentrations in massive copper alloys, after 2 h in gastric fluid, was only <0.01%-0.18%, consistent with the low surface reactivity of copper alloys (defined as 1 mm spheres). The average metal-specific migrations of cobalt, copper, nickel and lead from most of the tested copper alloys in gastric media are comparable to the ones from their pure metals. The data further show that the bioaccessibility of metals in massive copper alloys primarily depends on the bioelution medium, the exposed surface area and the composition of the alloy. The tested copper alloys show only limited evidence for influence of alloy surface microstructure. This is contrary to findings for other alloys such as stainless steel. Additional investigations on other copper alloys could allow to further refine these conclusions. These findings are useful for establishing the hazard and risk profile of copper alloys following oral exposure.

Estimating lung burdens based on individual particle density estimated from scanning electron microscopy and cascade impactor samples.

Workplace air is monitored for overall dust levels and for specific components of the dust to determine compliance with occupational and workplace standards established by regulatory bodies for worker health protection. Exposure monitoring studies were conducted by the International Copper Association (ICA) at various industrial facilities around the world working with copper. Individual cascade impactor stages were weighed to determine the total amount of dust collected on the stage, and then the amounts of soluble and insoluble copper and other metals on each stage were determined; speciation was not determined. Filter samples were also collected for scanning electron microscope analysis. Retrospectively, there was an interest in obtaining estimates of alveolar lung burdens of copper in workers engaged in tasks requiring different levels of exertion as reflected by their minute ventilation. However, mechanistic lung dosimetry models estimate alveolar lung burdens based on particle Stoke's diameter. In order to use these dosimetry models the mass-based, aerodynamic diameter distribution (which was measured) had to be transformed into a distribution of Stoke's diameters, requiring an estimation be made of individual particle density. This density value was estimated by using cascade impactor data together with scanning electron microscopy data from filter samples. The developed method was applied to ICA monitoring data sets and then the multiple path particle dosimetry (MPPD) model was used to determine the copper alveolar lung burdens for workers with different functional residual capacities engaged in activities requiring a range of minute ventilation levels.

Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells.

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family "zyklopen."

An exposure-response curve for copper excess and deficiency.

There is a need to define exposure-response curves for both Cu excess and deficiency to assist in determining the acceptable range of oral intake. A comprehensive database has been developed where different health outcomes from elevated and deficient Cu intakes were assigned ordinal severity scores to create common measures of response. A generalized linear model for ordinal data was used to estimate the probability of response associated with dose, duration and severity. The model can account for differences in animal species, the exposure medium (drinking water and feed), age, sex, and solubility. Using this model, an optimal intake level of 2.6 mg Cu/d was determined. This value is higher than the current U.S. recommended dietary intake (RDI; 0.9 mg/d) that protects against toxicity from Cu deficiency. It is also lower than the current tolerable upper intake level (UL; 10 mg/d) that protects against toxicity from Cu excess. Compared to traditional risk assessment approaches, categorical regression can provide risk managers with more information, including a range of intake levels associated with different levels of severity and probability of response. To weigh the relative harms of deficiency and excess, it is important that the results be interpreted along with the available information on the nature of the responses that were assigned to each severity score.

Risk assessment practice for essential metals.

This article addresses the content of the workshop, including a panel discussion relevant to delineation of a path forward in relation to risk assessment of essential metals. The state of the art of risk assessment and associated issues for essential metals are outlined initially, followed by brief illustration by the case studies considered at the workshop (i.e., copper, zinc, and manganese). Approaches for the future testing strategies of essential metals are discussed in terms of options to increase efficiency and accuracy of assessments. Subsequently, recommendations for pragmatic next steps to advance progress and facilitate uptake by the regulatory risk assessment community are presented.

Bioelution, Bioavailability, and Toxicity of Cobalt Compounds Correlate.

Based on the wide use of cobalt substances in a range of important technologies, it has become important to predict the toxicological properties of new or lesser-studied substances as accurately as possible. We studied a group of 6 cobalt substances with inorganic ligands, which were tested for their bioaccessibility (surrogate measure of bioavailability) through in vitro bioelution in simulated gastric and intestinal fluids. Representatives of the group also underwent in vivo blood kinetics and mass balance tests, and both oral acute and repeated dose toxicity (RDT) testing. We were able to show a good correlation between high in vitro bioaccessibility with high in vivo bioavailability and subsequent high in vivo toxicity; consequently, low in vitro bioaccessibility correlated well with low in vivo bioavailability and low in vivo toxicity. In vitro bioelution in simulated gastric fluid was the most precise predictor of the difference in the oral RDT lowest observed adverse effect levels of 2 compounds representing the highly and poorly bioaccessible subset of substances. The 2 compounds cobalt dichloride hexahydrate and tricobalt tetraoxide differed by a factor of 440 in their in vitro bioaccessibility and by a factor of 310 in their RDT lowest observed adverse effect level. In summary, this set of studies shows that solubility, specifically in vitro bioelution in simulated gastric fluid, is a good, yet conservative, predictor of in vivo bioavailability and oral systemic toxicity of inorganic cobalt substances. Bioelution data are therefore an invaluable tool for grouping and read across of cobalt substances for hazard and risk assessment.

Disturbed copper bioavailability in Alzheimer's disease.

Recent data from in vitro, animal, and human studies have shed new light on the positive roles of copper in many aspects of AD. Copper promotes the non-amyloidogenic processing of APP and thereby lowers the Aß production in cell culture systems, and it increases lifetime and decreases soluble amyloid production in APP transgenic mice. In a clinical trial with Alzheimer patients, the decline of Aß levels in CSF, which is a diagnostic marker, is diminished in the verum group (8 mg copper/day), indicating a beneficial effect of the copper treatment. These observations are in line with the benefit of treatment with compounds aimed at normalizing metal levels in the brain, such as PBT2. The data reviewed here demonstrate that there is an apparent disturbance in metal homeostasis in AD. More research is urgently needed to understand how this disturbance can be addressed therapeutically.

Cu loading alters expression of non-IRE regulated, but not IRE regulated, Fe dependent proteins in HepG2 cells.

This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using (125)I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.

Limited effects of glatiramer acetate in the high-copy number hSOD1-G93A mouse model of ALS.

In amyotrophic lateral sclerosis (ALS), an involvement of the immune system in the degenerative processes has been shown in both humans and the transgenic SOD1-G93A mice. We previously showed that Glatiramer acetate (also known as copolymer-1; COP-1; Copaxone) improves motor function and extends survival times in an inbred strain of ALS mice probably by interacting with pro-inflammatory T(H) lymphocytes. In the course of this study we tested whether these beneficial effects could be reproduced by repeated vaccination of animals with COP-1 without co-administration of complete Freund's adjuvant. In an outbred strain we could not demonstrate a positive effect of COP-1 on survival times, but found a significant improvement of motor performance during the late stage of disease and a moderate decrease of the production of the inflammatory cytokines interferon-gamma and IL-4 by T lymphocytes isolated from the mice's spleen. In conclusion, the effects of COP-1 in the applied hybrid strain displaying a faster disease progression were less pronounced than in the earlier tested inbred strain of ALS mice.

How reliable and robust are current biomarkers for copper status?

Cu is an essential nutrient for man, but can be toxic if intakes are too high. In sensitive populations, marginal over- or under-exposure can have detrimental effects. Malnourished children, the elderly, and pregnant or lactating females may be susceptible for Cu deficiency. Cu status and exposure in the population can currently not be easily measured, as neither plasma Cu nor plasma cuproenzymes reflect Cu status precisely. Some blood markers (such as ceruloplasmin) indicate severe Cu depletion, but do not inversely respond to Cu excess, and are not suitable to indicate marginal states. A biomarker of Cu is needed that is sensitive to small changes in Cu status, and that responds to Cu excess as well as deficiency. Such a marker will aid in monitoring Cu status in large populations, and will help to avoid chronic health effects (for example, liver damage in chronic toxicity, osteoporosis, loss of collagen stability, or increased susceptibility to infections in deficiency). The advent of high-throughput technologies has enabled us to screen for potential biomarkers in the whole proteome of a cell, not excluding markers that have no direct link to Cu. Further, this screening allows us to search for a whole group of proteins that, in combination, reflect Cu status. The present review emphasises the need to find sensitive biomarkers for Cu, examines potential markers of Cu status already available, and discusses methods to identify a novel suite of biomarkers.

Superoxide dismutase 1 modulates expression of transferrin receptor.

Copper-zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.

Placental ceruloplasmin homolog is regulated by iron and copper and is implicated in iron metabolism.

We previously reported an endogenous, membrane-bound Cu oxidase with homology to ceruloplasmin in BeWo cells, a placental choriocarcinoma cell line. In this previous study, ceruloplasmin immunoreactivity was localized to the perinuclear region and non-brush-border membranes. Here, we show that azide-sensitive oxidase activity is enriched in the same fractions, correlating subcellular localization of enzyme activity with ceruloplasmin immunoreactivity. Expression of the placental Cu oxidase is inversely proportional to Fe status and directly proportional to Cu status at enzyme and protein levels. To identify a role for the Cu oxidase, cells were exposed to (59)Fe-transferrin for 18 h in an environment of 20% O(2) or 5% O(2). At 5% O(2), Cu-deficient cells retain significantly more (59)Fe than control cells. This excess in (59)Fe accumulation is caused by a significant decrease in (59)Fe release. These results indicate that downregulation of the placental Cu oxidase in BeWo cells impairs Fe release. This effect is only apparent in an environment of limited O(2).

Iron and copper interactions in development and the effect on pregnancy outcome.

During pregnancy, nutrients are transferred from mother to fetus across the placenta. The mechanisms whereby this occurs, and the adaptations that occur in response to deficiency or overload of iron (Fe) and copper (Cu) are examined in this review. Fe deficiency during pregnancy is common and has serious consequences both in the short and the long term such as fetal growth retardation and cardiovascular problems in the adult offspring. Similarly, Cu deficiency, although not so common, also has deleterious effects. The placenta minimizes the effect of the deficiency by up-regulating the proteins involved in Fe transfer. For example, transferrin receptor levels increase inversely to maternal Fe levels. Divalent metal transporter 1 (DMT1) mRNA in the iron-responsive element (IRE) regulated, but not the non-IRE regulated form is increased, as is the placenta Cu oxidase. Conversely, iron-regulated gene 1 (IREG1) expression is not affected. Fe deficiency increases Cu levels in maternal liver, serum and placenta, but has much less effect in the fetal serum and liver. Apart from maternal ceruloplasmin, mRNA levels of Cu-related proteins are not changed. The Cu oxidase, which we suggest fulfils the function of hephaestin in placenta, is regulated by Cu as well as by Fe. Fe deficiency also has marked effects on cytokine levels in the placenta. Tumor necrosis factor alpha (TNFalpha) and TNFalpha receptor 1 (TNFalphaR1) levels both increase. The data show that altering Fe status has a marked effect on metabolism of other metals and of other important mediators of cell function. This is particularly important during pregnancy, when the developing fetus is very vulnerable to inappropriate micronutrient status.