BACKGROUND
in the united states, more than a million workers are potentially exposed to cobalt or its compounds  <cit> .
cobalt is massively used in the steel industry, being a major constituent of hard metal alloys, in combination with tungsten carbides.
other industrial uses include diamond polishing with co-containing disks and the production of drying agents, pigments, and catalysts  <cit> .
radioactive isotopes of cobalt are used in industry, medicine and nuclear research.
in nuclear power plants, 59co-containing alloys can be activated into radioactive 60co oxides, dispersed in the cooling water and then contaminate workers  <cit> .
a study measuring the ambient air in cobalt powder production reported concentrations of cobalt ranging from  <dig>  to  <dig> mg/m <dig>  <cit> .
airborne concentrations measured in the working environment from a factory producing hard-metal inserts ranged from  <dig>  to  <dig>  mg/m <dig>  <cit> .
occupational exposure to co occurs mainly via inhalation leading to various lung diseases, such as pneumonitis, fibrosis and asthma  <cit> .
as with human exposure, animal exposure to cobalt-containing aerosols causes pronounced respiratory effects.
a single 30-minute exposure of rats to relatively high levels , resulted in lung congestion, oedema, and haemorrhage  <cit> .
necrosis and inflammation of the respiratory tract epithelium were reported in rats exposed to  <dig> mg cobalt/m <dig> and mice exposed to  <dig>  mg cobalt sulfate/m <dig> over  <dig> days  <cit> .
some acute effects have been observed concerning general public exposure.
lethal cardiomyopathy was reported in people who consumed large quantities of beer containing cobalt as a foam stabilizer , and acute mortality accounted for 18% of the deaths  <cit> .
a 19-month-old boy who swallowed an unknown amount of cobalt chloride solution died  <dig>  hours after ingestion  <cit> .
following absorbtion by inhalation, cobalt is eliminated in the urine.
biological monitoring of accidental exposure mainly involves measuring the concentration of metal in the urine.
this might be inadequate for several reasons.
firstly, the quantity of metal excreted  does not necessarily reflect organ damage, which varies from one person to another.
secondly, depending on the chemical form, excretion does not necessarily reflect the level of metal in the body because some forms are retained in the lungs.
thirdly, depending on its solubility, clearance can be very rapid and the cobalt may have left the body by the time samples can be taken.
therefore, a key issue in monitoring occupational exposure is the availability of adequate biomarkers.
although the chemical toxicity of cobalt has been proven, the molecular mechanisms of its toxicity are not all known.
cobalt is genotoxic  <cit> , and an oxidizing stress inducer  <cit> .
it also induces apoptosis  <cit> .
cobalt is used as a hypoxia-simulating agent  <cit> , leading to increased apoptosis, glycolysis, angiogenesis and erythropoiesis <cit> .
since the lung is the main target organ of cobalt toxicity, the human a <dig> lung cell line was chosen as a model for this study, to evaluate cobalt toxicity.
noteworthy, this cell line has been widely documented in molecular toxicology, including hypoxia mimicked by cobalt  <cit> .
microarrays are currently used for large scale gene profiling, measuring sensitive cell changes in response to xenobiotic exposure.
such investigative studies may help identify new molecular targets for toxicants or provide new hypotheses about their mechanisms of action  <cit> .
we used toxicogenomic tools to detect biomolecular targets of acute cobalt exposure and identify candidates as biomarkers of cobalt toxicity.
RESULTS
cobalt cytotoxicity
a <dig> is a stable tumor cell line, obtained from human lung carcinoma, with properties of type ii alveolar epithelial cells  <cit> .
the response of a <dig> cells to increasing concentrations of cobalt  after  <dig> h exposure was first analysed using the intracellular atp measurement that reflects early metabolic modification  <cit> .
we determined that the concentration needed to decrease atp concentration to 50% was reached at  <dig> mm cobalt .
at this concentration, we measured an average load of  <dig>  pg of cobalt per cell, using flame atomic absorption spectroscopy .
some toxicologists, including one reviewer of this paper, assume that cytotoxic doses are too high to study specific cellular response to a toxicant  <cit> .
the consequence, on gene modulation, could be the activation of large numbers of nonspecific pathways of toxicity, particularly a predominance of apoptosis and stress related genes.
however, as the purpose of this study was to simulate accidental acute exposure, we nevertheless chose to expose a <dig> cells to an acute dose of cobalt .
differential gene expression and functional classification
the global transcriptional response was monitored using cea microarrays   <cit> .
a <dig> cells were treated with  <dig> mm cobalt in a time course experiment.
for each experiment, control cells were grown in parallel and collected simultaneously.
at  <dig> min.
and  <dig> h, six microarrays  were hybridized with rna from two different cell exposures.
at  <dig> h, four microarrays  were hybridized with rna from two exposures and at  <dig> h,  <dig> microarrays  were hybridized with rna from three cell exposures.
details of the experimental design and microarray data were submitted to the gene expression omnibus repository at the national center for biotechnology information  <cit>  and are accessible via number gse <dig>  data analysis was performed as described in the methods section.
after applying our selection criteria to the data , we obtained a list of  <dig> modulated clones corresponding to  <dig> known genes.
the modulation trend was up-regulation since  <dig> genes were up-regulated and  <dig> were down-regulated .
the results showed that the main effect was obtained after  <dig> h of cobalt treatment , and to a lesser extent after  <dig> min .
at  <dig> h and  <dig> h,  <dig> and  <dig> genes respectively were found to be modulated.
since  <dig> h was the main category in terms of modulated gene numbers, we reinforced the study for this time point by performing a total of  <dig> microarrays.
dnaja2
oaz1
pgf
st13
tnfrsf9
dlg1
gnb2l1
slc12a5
slc25a6
slc2a1
bnip3l
ifna4
serpinc1
hspcb
ubc
bhc80
hoxa9
ltbp3
a: p value <  <dig>  with  <dig>  fold change.
b: validation with quantitative qrt-pcr  :
*: p value <  <dig> , ** p value <  <dig> , *** p value <  <dig> .
microarray p-values were calculated using a t-test statistical analysis  on genespring software.
pair wise fixed reallocation randomized test was used to calculate qrt-pcr p values with rest software.
ndrg4
axl
gfra2
ifnar2
mapk10
tfrc
bag1
fbxl2
timp2
maz
sin3a
a: p value <  <dig>  with  <dig>  fold change.
b: validation with quantitative qrt-pcr  :
*: p value <  <dig> , ** p value <  <dig> , *** p value <  <dig> , **** p value <  <dig> .
microarray p-values were calculated using a t-test statistical analysis  on genespring software.
pair wise fixed reallocation randomized test was used to calculate qrt-pcr p values with rest software.
we used david  <cit>  web-accessible software, to obtain information from the gene ontology database.
we then sorted the modulated genes manually into  <dig> major functional classes , combining information from david, swiss-prot and additional elements taken from the literature.
the classifications of genes modulated after  <dig> min and  <dig> h of cobalt treatment were compared.
at  <dig> min, the main category regulated was signal transduction and trafficking  and half of the modulated genes coded for membrane proteins.
this suggests strong involvement of membrane transport in the early response to cobalt exposure.
the later response  mainly concerned protein expression and turnover , metabolism and energy , and finally signal transduction and trafficking .
this different pattern indicates that after  <dig> h, the cell response is established, with metabolic adaptation and strong involvement of protein translation.
it can be noticed that in spite of the cytotoxic dose we used, the genes modulation trend was rather increase than decrease, indicating an active metabolic response of the cell, and also that apoptotic and stress related genes are not over represented.
quantitative rt-pcr
we selected genes to be validated by quantitative rt-pcr  in some specific functional classes: development, differentiation and proliferation, signal transduction and trafficking, cell defense and finally gene transcription and modification.
we also selected genes coding for secreted proteins as potential hits for biomarker research.
tables  <dig> and  <dig> show the results of quantitative rt-pcr validation  and the primers used are described in table  <dig>  of the  <dig> genes tested, most confirmed the variation observed in the microarrays .
five genes appeared to be modulated in the opposite direction by qrt-pcr .
some discrepancies between the two techniques may be due to technical artefacts even if the primers were designed within the microarray cdna sequence.
for further analysis of gene modulation, we performed time-course quantitative rt-pcr on several genes: axl, ubc, fbxl <dig> and slc12a <dig> .
the results show that modulation varies over time following various profiles: either diminishing at first, then increasing up to  <dig> h , or being induced from  <dig> h , or finally, early and continuous diminishing .
for genes displaying temporal modulation, down-fluctuation quickly followed by up-regulation, as with axl , a slight difference in the kinetics of different exposures could explain the opposing results we found between microarray and qrt-pcr ratios, because rnas from different exposures were used to perform the microarray experiments and qrt-pcr.
these results, revealing an oscillatory gene expression profile, suggest complex regulation pathways in response to cobalt.
hypoxia
cobalt is known as a "hypoxia-simulating" agent <cit>  and most of the literature therefore relates to this property.
hypoxia-inducible factor  is a transcription factor which acts as the main regulator of oxygen homeostasis and the genes it induces have mostly been described  <cit> .
we compared this list with our results to identify modulations due to hypoxia.
strikingly, very poor overlap was observed, as only  <dig> of the  <dig> modulated genes  in our study matched hif <dig> target genes.
these  <dig> genes are: aldolase a , glucose transporter  <dig> , glyceraldehyde 3-phosphate dehydrogenase , lactate dehydrogenase a , proapoptotic factor , phosphoglycerate kinase  <dig>  and transferrin receptor , .
like hif <dig> regulated genes, they were all induced at  <dig> h by cobalt , therefore our results agree with those already published and validate our system relative to previous reports, since they represent an appropriate internal positive control.
recently, two transcriptomic studies of hypoxia compared the action of low oxygen with that of metal ions such as cobalt or nickel on embryonic mouse fibrobasts  <cit>  and human liver carcinoma  <cit> .
21% and 28% of the genes respectively were found to be modulated by both hypoxia and cobalt.
this shows that the toxic effect of cobalt is not limited to hypoxia.
it should also be noted that nickel had an action closer to that of low oxygen than cobalt, with 65% common modulated genes  <cit> .
comparing these results with ours showed that, apart from the hypoxia genes, we had no overlap.
this discrepancy might be due to differences between cell models, species and conditions of exposure .
stress response – apoptosis
the hspcb gene coding for heat shock protein  <dig>  was induced by a factor of  <dig>  at  <dig> h .
hsp <dig> protein plays a major role in the stress response by preventing irreversible protein aggregation.
in our previous study of the toxicogenomics of uranium  <cit> , we noted that both hspcb gene and hsp <dig> protein were strongly repressed.
this protein can even be thought of as a metal "sensor", since the hspcb gene was also reported to be modulated by other metals   <cit> .
however, we did not see any change in the quantity of hsp <dig> in western blotting experiments .
this proteome/transcriptome discordance can be explained by an increase in protein turnover associated with gene induction, resulting in a constant amount of protein being maintained in the cell.
another explanation may be that, as gerner observed for apoptosis, hsp <dig> is aggregated and accumulates in the insoluble fraction of the cytosol and is not collected during protein extraction  <cit> .
two other genes described as hsp mediators are also induced by cobalt: st <dig> and dnaja <dig>
ubiquitin is a key element in eliminating proteins via proteasome because polyubiquitinylation is the recognition signal for protein elimination  <cit> .
the results show that ubiquitin genes were up-regulated after  <dig> h of toxic exposure  while ubc gene induction was observed in the kinetics from  <dig> h with qrt-pcr , showing that it is not an early event in cell response.
the western blot revealed protein ubiquitinylation, with the anti-ubiquitin antibody, shown by a smear at high molecular weight in the cobalt extract, indicating an accumulation of ubiquitinylated protein .
this demonstrates increased turnover of proteins involved in defense against stress, or the elimination of proteins partly damaged by the metal, or the start of apoptosis.
cobalt has been described as an apoptosis inducer  <cit>  and this is confirmed here by bag <dig> repression and bnip3l induction .
tumor suppressors – transcription factors
the protein encoded by maz , is a transcription factor involved in both the initiation and termination of target gene transcription.
it binds zinc and can act as a tumor suppressor  <cit> .
this gene is repressed early at  <dig> h. other cobalt-modulated genes code for zinc-binding proteins involved in transcription or dna metabolism: bhc <dig> , nt5e  and sdccag <dig> .
cobalt ions have been shown to substitute for zinc in zinc finger protein domains which control the transcription of several genes and also in zinc-dna repair proteins, inhibiting dna repair  <cit> .
dlg <dig>  has been identified as a tumor suppressor gene in drosophila, with its mutation inducing the loss of cell polarity and neoplastic tissue growth.
this gene is highly conserved.
the molecular mechanism which regulates cell proliferation by dlg <dig> is not well known but there are arguments for implicating epithelial cell polarization in this regulation  <cit> .
the dlg <dig>  gene was strongly repressed in qrt-pcr.
the anti-dlg <dig> antibody revealed clear depletion of dlg <dig> protein in co+ samples .
this confirmed the results of the qrt-pcr.
two tumor suppressors  were repressed by cobalt, and in one case , this repression was also observed at protein level.
we noted the modulation of three genes related to myc, a key element in oncogenic response .
the first, sin3a, known as an myc suppressor, is induced by cobalt.
the two others: ndrg <dig>  a gene negatively regulated by myc and maz, a myc transcription inhibitor, are down-regulated by cobalt.
these results are not clear regarding myc up- or down-regulation, but myc regulation pathways are complex.
these results led us to hypothesize that myc might be modulated by cobalt, so we tested myc temporal modulation with qrt-pcr.
interestingly, we noted significant myc inhibition from  <dig> min to  <dig> h  indicating that cobalt modulates regulation pathways controlling or controlled by myc.
liu has recently shown that myc is involved in the carcinogenic response of mouse liver to arsenic, another toxic metal  <cit> .
the axl gene is positively modulated.
members of the axl/ufo family of tyrosine kinases are prone to transcriptional regulation and perform various functions including regulation of cell adhesion, migration, phagocytosis, and survival.
the biological consequences of axl activation are complex.
axl was initially identified as a transforming gene product, and axl expression is indeed up-regulated in human tumors  <cit> .
concerning the potential effects of cobalt on cancer development, it will be interesting to carry out further studies of the mechanisms by which cobalt induces these responses.
these genes have never been linked to cobalt effects before.
transporters
two cobalt transporters are described in yeast: cot <dig> and cot <dig>  <cit> .
eleven cobalt-tolerant mutants were obtained with a common cot <dig> mutation, suggesting that it is involved in cobalt uptake  <cit> .
the mutants were resistant to cobalt toxicity and cobalt was not incorporated in yeast cells.
the human homolog of cot <dig>  fbxl <dig>  was strongly repressed in our study as early as  <dig> h and with a ratio of  <dig>  at  <dig> h; this is the strongest inhibition observed in time-course qrt-pcr analysis .
fbxl <dig> function is not very well described, but it is potentially involved in ubiquitinylation.
conklin's observations on the yeast homolog of fbxl <dig>  <cit>  and our results, lead us to suggest that fbxl <dig> gene repression could be a cellular defense mechanism and this protein might be associated with cobalt uptake in humans.
the product of the cot <dig> gene is involved in the uptake of cobalt ions  <cit> .
cot <dig>  has a human homolog in znt <dig>  coding for zinc transporter  <dig> protein, probably involved in zinc transport out of the cell.
znt <dig>  not spotted on the microarray, was also tested in kinetics with qrt-pcr and was found to be slightly modulated with a biphasic response; first down- then up-regulated .
the slc12a <dig> gene, coding for an integral membrane k-cl cotransporter, was repressed at  <dig> h then strongly increased at  <dig> h .
the three genes, fbxl <dig>  znt <dig> and slc12a <dig>  are therefore appropriate candidates for research into cobalt transport proteins in human cells and will be studied further using targeted biological approaches.
potential biomarkers and secreted proteins
biological monitoring of exposure to cobalt is mainly based on its concentration in urine.
this method may be inadequate for two reasons.
firstly, measuring the quantity of excreted metal  does not necessarily reflect organ damage, which varies from one person to another.
furthermore, depending on the chemical form, excretion does not necessarily reflect the concentration of metal in the body; indeed, certain forms are eliminated in several phases; only 40% of cobalt oxides inhaled are excreted within  <dig> h  <cit> .
in this context, only an effective biomarker could evaluate the damage and provide a valuable tool for monitoring occupational contamination in the event of accidental acute exposure or chronic exposure.
we selected nine modulated genes coding for secreted proteins: icam <dig> , serpin c <dig> , ifna <dig> , tfrc , pla2r <dig> , tnfsf <dig> , wfdc <dig> , ltbp <dig>  and timp <dig> .
to further evaluate the interest of these genes, we checked the secretion modulation of the corresponding proteins.
a <dig> cells were exposed to cobalt in a dose-response manner, to  <dig> ,  <dig> and  <dig> mm, and the supernatants analysed using available immunoassays.
the levels of fas ligand, wfdc <dig>  <cit> , icam <dig>  antithrombin-iii, alpha- <dig> interferon, and soluble transferrin receptor were found to be invariant or below the kit detection limit.
these results indicate that most of these immunoassays were not sensitive enough to detect faint modulations.
timp <dig> protein  was measured using elisa and we confirmed the modulation recorded at gene level .
a strong decrease in timp <dig> supernatant concentration was noted at  <dig> or  <dig> mm of cobalt .
the changes in timp <dig> levels in cell supernatants between cobalt and the control were -5% for  <dig>  mm, -57% for  <dig> mm and -72% for  <dig> mm.
the reason for this reduction in timp <dig> can be explained as a consequence of cobalt exposure.
matrix metalloproteinases  are a family of endopeptidases that can degrade all the components of the extracellular matrix  <cit> .
endogenous protease inhibitors, known as tissue inhibitors of metalloproteinase , provide critical extracellular regulation of mmp proteolytic activities  <cit> , regulating tumor growth, progression, and angiogenesis in a variety of experimental cancer models and human malignancies.
timp <dig> mrna is described as being suppressed by hypoxia in endothelial cells  <cit> .
exposure of cells to cobalt up-regulates indirectly hypoxia-inducible genes by a mechanism which has recently been explained  <cit> .
therefore, timp- <dig> down-regulation might be a consequence of cobalt-simulated hypoxia.
down-regulation of timp <dig> mediated by ros production has been observed by rezzani in rat cardiac tissue exposed to cyclosporin  <cit> .
since it is known that cobalt triggers ros production  <cit> , timp <dig> modulation by cobalt could also be explained by this pathway.
timp <dig> protein modulation in cobalt-exposed cell supernatant corroborated the gene down-regulation observed on the microarrays and with qrt-pcr.
timp <dig> modulation has never been associated with metal stress.
this result is innovative and will be studied further in the biological fluids of rats exposed to different chemical forms of cobalt by inhalation.
CONCLUSIONS
this study provides the first toxicogenomic analysis of human lung cell response to acute cobalt exposure.
we have confirmed that genes involved in the cobalt hypoxia response and apoptosis are modulated.
we have also revealed genes linked to heat shock response and proteasome function that have already been described in other metal stress responses.
newly identified genes linked to cobalt acute toxicity include potential cobalt carriers  and tumor suppressors or transcription factors .
some of these genes provide new hypotheses for elucidating the mechanisms of cobalt intracellular chemical toxicity.
targeted biological approaches might confirm their biochemical role in the cobalt response.
regarding biomarkers, we have highlighted the down-regulation of timp <dig>  a gene coding for a secreted protein.
timp <dig> modulation was confirmed at protein level, in a dose-dependent manner, in the supernatant of exposed a <dig> cells.
timp <dig> provides a putative biomarker of cobalt toxicity that will be further studied on animal models.

 in the united states, more than a million workers are potentially exposed to cobalt or its compounds. cobalt is massively used in the steel industry, being a major constituent of hard metal alloys, in combination with tungsten carbides. other industrial uses include diamond polishing with co-containing disks and the production of drying agents, pigments, and catalysts. radioactive isotopes of cobalt are used in industry, medicine and nuclear research. in nuclear power plants, 59co-containing alloys can be activated into radioactive 60co oxides, dispersed in the cooling water and then contaminate workers. a study measuring the ambient air in cobalt powder production reported concentrations of cobalt ranging from to mg/m. airborne concentrations measured in the working environment from a factory producing hard-metal inserts ranged from to mg/m. occupational exposure to co occurs mainly via inhalation leading to various lung diseases, such as pneumonitis, fibrosis and asthma. as with human exposure, animal exposure to cobalt-containing aerosols causes pronounced respiratory effects. a single 30-minute exposure of rats to relatively high levels, resulted in lung congestion, oedema, and haemorrhage. necrosis and inflammation of the respiratory tract epithelium were reported in rats exposed to mg cobalt/m and mice exposed to mg cobalt sulfate/m over days. some acute effects have been observed concerning general public exposure. lethal cardiomyopathy was reported in people who consumed large quantities of beer containing cobalt as a foam stabilizer, and acute mortality accounted for 18% of the deaths. a 19-month-old boy who swallowed an unknown amount of cobalt chloride solution died hours after ingestion. following absorbtion by inhalation, cobalt is eliminated in the urine. biological monitoring of accidental exposure mainly involves measuring the concentration of metal in the urine. this might be inadequate for several reasons. firstly, the quantity of metal excreted does not necessarily reflect organ damage, which varies from one person to another. secondly, depending on the chemical form, excretion does not necessarily reflect the level of metal in the body because some forms are retained in the lungs. thirdly, depending on its solubility, clearance can be very rapid and the cobalt may have left the body by the time samples can be taken. therefore, a key issue in monitoring occupational exposure is the availability of adequate biomarkers. although the chemical toxicity of cobalt has been proven, the molecular mechanisms of its toxicity are not all known. cobalt is genotoxic, and an oxidizing stress inducer. it also induces apoptosis. cobalt is used as a hypoxia-simulating agent, leading to increased apoptosis, glycolysis, angiogenesis and erythropoiesis. since the lung is the main target organ of cobalt toxicity, the human a lung cell line was chosen as a model for this study, to evaluate cobalt toxicity. noteworthy, this cell line has been widely documented in molecular toxicology, including hypoxia mimicked by cobalt. microarrays are currently used for large scale gene profiling, measuring sensitive cell changes in response to xenobiotic exposure. such investigative studies may help identify new molecular targets for toxicants or provide new hypotheses about their mechanisms of action. we used toxicogenomic tools to detect biomolecular targets of acute cobalt exposure and identify candidates as biomarkers of cobalt toxicity.  cobalt cytotoxicity a is a stable tumor cell line, obtained from human lung carcinoma, with properties of type ii alveolar epithelial cells. the response of a cells to increasing concentrations of cobalt after h exposure was first analysed using the intracellular atp measurement that reflects early metabolic modification. we determined that the concentration needed to decrease atp concentration to 50% was reached at mm cobalt. at this concentration, we measured an average load of pg of cobalt per cell, using flame atomic absorption spectroscopy. some toxicologists, including one reviewer of this paper, assume that cytotoxic doses are too high to study specific cellular response to a toxicant. the consequence, on gene modulation, could be the activation of large numbers of nonspecific pathways of toxicity, particularly a predominance of apoptosis and stress related genes. however, as the purpose of this study was to simulate accidental acute exposure, we nevertheless chose to expose a cells to an acute dose of cobalt. differential gene expression and functional classification the global transcriptional response was monitored using cea microarrays. a cells were treated with mm cobalt in a time course experiment. for each experiment, control cells were grown in parallel and collected simultaneously. at min. and h, six microarrays were hybridized with rna from two different cell exposures. at h, four microarrays were hybridized with rna from two exposures and at h, microarrays were hybridized with rna from three cell exposures. details of the experimental design and microarray data were submitted to the gene expression omnibus repository at the national center for biotechnology information and are accessible via number gse data analysis was performed as described in the methods section. after applying our selection criteria to the data, we obtained a list of modulated clones corresponding to known genes. the modulation trend was up-regulation since genes were up-regulated and were down-regulated. the results showed that the main effect was obtained after h of cobalt treatment, and to a lesser extent after min. at h and h, and genes respectively were found to be modulated. since h was the main category in terms of modulated gene numbers, we reinforced the study for this time point by performing a total of microarrays. dnaja2 oaz1 pgf st13 tnfrsf9 dlg1 gnb2l1 slc12a5 slc25a6 slc2a1 bnip3l ifna4 serpinc1 hspcb ubc bhc80 hoxa9 ltbp3 a: p value < with fold change. b: validation with quantitative qrt-pcr: *: p value <, ** p value <, *** p value <. microarray p-values were calculated using a t-test statistical analysis on genespring software. pair wise fixed reallocation randomized test was used to calculate qrt-pcr p values with rest software. ndrg4 axl gfra2 ifnar2 mapk10 tfrc bag1 fbxl2 timp2 maz sin3a a: p value < with fold change. b: validation with quantitative qrt-pcr: *: p value <, ** p value <, *** p value <, **** p value <. microarray p-values were calculated using a t-test statistical analysis on genespring software. pair wise fixed reallocation randomized test was used to calculate qrt-pcr p values with rest software. we used david web-accessible software, to obtain information from the gene ontology database. we then sorted the modulated genes manually into major functional classes, combining information from david, swiss-prot and additional elements taken from the literature. the classifications of genes modulated after min and h of cobalt treatment were compared. at min, the main category regulated was signal transduction and trafficking and half of the modulated genes coded for membrane proteins. this suggests strong involvement of membrane transport in the early response to cobalt exposure. the later response mainly concerned protein expression and turnover, metabolism and energy, and finally signal transduction and trafficking. this different pattern indicates that after h, the cell response is established, with metabolic adaptation and strong involvement of protein translation. it can be noticed that in spite of the cytotoxic dose we used, the genes modulation trend was rather increase than decrease, indicating an active metabolic response of the cell, and also that apoptotic and stress related genes are not over represented. quantitative rt-pcr we selected genes to be validated by quantitative rt-pcr in some specific functional classes: development, differentiation and proliferation, signal transduction and trafficking, cell defense and finally gene transcription and modification. we also selected genes coding for secreted proteins as potential hits for biomarker research. tables and show the results of quantitative rt-pcr validation and the primers used are described in table of the genes tested, most confirmed the variation observed in the microarrays. five genes appeared to be modulated in the opposite direction by qrt-pcr. some discrepancies between the two techniques may be due to technical artefacts even if the primers were designed within the microarray cdna sequence. for further analysis of gene modulation, we performed time-course quantitative rt-pcr on several genes: axl, ubc, fbxl and slc12a. the results show that modulation varies over time following various profiles: either diminishing at first, then increasing up to h, or being induced from h, or finally, early and continuous diminishing. for genes displaying temporal modulation, down-fluctuation quickly followed by up-regulation, as with axl, a slight difference in the kinetics of different exposures could explain the opposing results we found between microarray and qrt-pcr ratios, because rnas from different exposures were used to perform the microarray experiments and qrt-pcr. these results, revealing an oscillatory gene expression profile, suggest complex regulation pathways in response to cobalt. hypoxia cobalt is known as a "hypoxia-simulating" agent and most of the literature therefore relates to this property. hypoxia-inducible factor is a transcription factor which acts as the main regulator of oxygen homeostasis and the genes it induces have mostly been described. we compared this list with our results to identify modulations due to hypoxia. strikingly, very poor overlap was observed, as only of the modulated genes in our study matched hif target genes. these genes are: aldolase a, glucose transporter, glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase a, proapoptotic factor, phosphoglycerate kinase and transferrin receptor,. like hif regulated genes, they were all induced at h by cobalt, therefore our results agree with those already published and validate our system relative to previous reports, since they represent an appropriate internal positive control. recently, two transcriptomic studies of hypoxia compared the action of low oxygen with that of metal ions such as cobalt or nickel on embryonic mouse fibrobasts and human liver carcinoma. 21% and 28% of the genes respectively were found to be modulated by both hypoxia and cobalt. this shows that the toxic effect of cobalt is not limited to hypoxia. it should also be noted that nickel had an action closer to that of low oxygen than cobalt, with 65% common modulated genes. comparing these results with ours showed that, apart from the hypoxia genes, we had no overlap. this discrepancy might be due to differences between cell models, species and conditions of exposure. stress response – apoptosis the hspcb gene coding for heat shock protein was induced by a factor of at h. hsp protein plays a major role in the stress response by preventing irreversible protein aggregation. in our previous study of the toxicogenomics of uranium, we noted that both hspcb gene and hsp protein were strongly repressed. this protein can even be thought of as a metal "sensor", since the hspcb gene was also reported to be modulated by other metals. however, we did not see any change in the quantity of hsp in western blotting experiments. this proteome/transcriptome discordance can be explained by an increase in protein turnover associated with gene induction, resulting in a constant amount of protein being maintained in the cell. another explanation may be that, as gerner observed for apoptosis, hsp is aggregated and accumulates in the insoluble fraction of the cytosol and is not collected during protein extraction. two other genes described as hsp mediators are also induced by cobalt: st and dnaja ubiquitin is a key element in eliminating proteins via proteasome because polyubiquitinylation is the recognition signal for protein elimination. the results show that ubiquitin genes were up-regulated after h of toxic exposure while ubc gene induction was observed in the kinetics from h with qrt-pcr, showing that it is not an early event in cell response. the western blot revealed protein ubiquitinylation, with the anti-ubiquitin antibody, shown by a smear at high molecular weight in the cobalt extract, indicating an accumulation of ubiquitinylated protein. this demonstrates increased turnover of proteins involved in defense against stress, or the elimination of proteins partly damaged by the metal, or the start of apoptosis. cobalt has been described as an apoptosis inducer and this is confirmed here by bag repression and bnip3l induction. tumor suppressors – transcription factors the protein encoded by maz, is a transcription factor involved in both the initiation and termination of target gene transcription. it binds zinc and can act as a tumor suppressor. this gene is repressed early at h. other cobalt-modulated genes code for zinc-binding proteins involved in transcription or dna metabolism: bhc, nt5e and sdccag. cobalt ions have been shown to substitute for zinc in zinc finger protein domains which control the transcription of several genes and also in zinc-dna repair proteins, inhibiting dna repair. dlg has been identified as a tumor suppressor gene in drosophila, with its mutation inducing the loss of cell polarity and neoplastic tissue growth. this gene is highly conserved. the molecular mechanism which regulates cell proliferation by dlg is not well known but there are arguments for implicating epithelial cell polarization in this regulation. the dlg gene was strongly repressed in qrt-pcr. the anti-dlg antibody revealed clear depletion of dlg protein in co+ samples. this confirmed the results of the qrt-pcr. two tumor suppressors were repressed by cobalt, and in one case, this repression was also observed at protein level. we noted the modulation of three genes related to myc, a key element in oncogenic response. the first, sin3a, known as an myc suppressor, is induced by cobalt. the two others: ndrg a gene negatively regulated by myc and maz, a myc transcription inhibitor, are down-regulated by cobalt. these results are not clear regarding myc up- or down-regulation, but myc regulation pathways are complex. these results led us to hypothesize that myc might be modulated by cobalt, so we tested myc temporal modulation with qrt-pcr. interestingly, we noted significant myc inhibition from min to h indicating that cobalt modulates regulation pathways controlling or controlled by myc. liu has recently shown that myc is involved in the carcinogenic response of mouse liver to arsenic, another toxic metal. the axl gene is positively modulated. members of the axl/ufo family of tyrosine kinases are prone to transcriptional regulation and perform various functions including regulation of cell adhesion, migration, phagocytosis, and survival. the biological consequences of axl activation are complex. axl was initially identified as a transforming gene product, and axl expression is indeed up-regulated in human tumors. concerning the potential effects of cobalt on cancer development, it will be interesting to carry out further studies of the mechanisms by which cobalt induces these responses. these genes have never been linked to cobalt effects before. transporters two cobalt transporters are described in yeast: cot and cot. eleven cobalt-tolerant mutants were obtained with a common cot mutation, suggesting that it is involved in cobalt uptake. the mutants were resistant to cobalt toxicity and cobalt was not incorporated in yeast cells. the human homolog of cot fbxl was strongly repressed in our study as early as h and with a ratio of at h; this is the strongest inhibition observed in time-course qrt-pcr analysis. fbxl function is not very well described, but it is potentially involved in ubiquitinylation. conklin's observations on the yeast homolog of fbxl and our results, lead us to suggest that fbxl gene repression could be a cellular defense mechanism and this protein might be associated with cobalt uptake in humans. the product of the cot gene is involved in the uptake of cobalt ions. cot has a human homolog in znt coding for zinc transporter protein, probably involved in zinc transport out of the cell. znt not spotted on the microarray, was also tested in kinetics with qrt-pcr and was found to be slightly modulated with a biphasic response; first down- then up-regulated. the slc12a gene, coding for an integral membrane k-cl cotransporter, was repressed at h then strongly increased at h. the three genes, fbxl znt and slc12a are therefore appropriate candidates for research into cobalt transport proteins in human cells and will be studied further using targeted biological approaches. potential biomarkers and secreted proteins biological monitoring of exposure to cobalt is mainly based on its concentration in urine. this method may be inadequate for two reasons. firstly, measuring the quantity of excreted metal does not necessarily reflect organ damage, which varies from one person to another. furthermore, depending on the chemical form, excretion does not necessarily reflect the concentration of metal in the body; indeed, certain forms are eliminated in several phases; only 40% of cobalt oxides inhaled are excreted within h. in this context, only an effective biomarker could evaluate the damage and provide a valuable tool for monitoring occupational contamination in the event of accidental acute exposure or chronic exposure. we selected nine modulated genes coding for secreted proteins: icam, serpin c, ifna, tfrc, pla2r, tnfsf, wfdc, ltbp and timp. to further evaluate the interest of these genes, we checked the secretion modulation of the corresponding proteins. a cells were exposed to cobalt in a dose-response manner, to, and mm, and the supernatants analysed using available immunoassays. the levels of fas ligand, wfdc, icam antithrombin-iii, alpha- interferon, and soluble transferrin receptor were found to be invariant or below the kit detection limit. these results indicate that most of these immunoassays were not sensitive enough to detect faint modulations. timp protein was measured using elisa and we confirmed the modulation recorded at gene level. a strong decrease in timp supernatant concentration was noted at or mm of cobalt. the changes in timp levels in cell supernatants between cobalt and the control were -5% for mm, -57% for mm and -72% for mm. the reason for this reduction in timp can be explained as a consequence of cobalt exposure. matrix metalloproteinases are a family of endopeptidases that can degrade all the components of the extracellular matrix. endogenous protease inhibitors, known as tissue inhibitors of metalloproteinase, provide critical extracellular regulation of mmp proteolytic activities, regulating tumor growth, progression, and angiogenesis in a variety of experimental cancer models and human malignancies. timp mrna is described as being suppressed by hypoxia in endothelial cells. exposure of cells to cobalt up-regulates indirectly hypoxia-inducible genes by a mechanism which has recently been explained. therefore, timp- down-regulation might be a consequence of cobalt-simulated hypoxia. down-regulation of timp mediated by ros production has been observed by rezzani in rat cardiac tissue exposed to cyclosporin. since it is known that cobalt triggers ros production, timp modulation by cobalt could also be explained by this pathway. timp protein modulation in cobalt-exposed cell supernatant corroborated the gene down-regulation observed on the microarrays and with qrt-pcr. timp modulation has never been associated with metal stress. this result is innovative and will be studied further in the biological fluids of rats exposed to different chemical forms of cobalt by inhalation.  this study provides the first toxicogenomic analysis of human lung cell response to acute cobalt exposure. we have confirmed that genes involved in the cobalt hypoxia response and apoptosis are modulated. we have also revealed genes linked to heat shock response and proteasome function that have already been described in other metal stress responses. newly identified genes linked to cobalt acute toxicity include potential cobalt carriers and tumor suppressors or transcription factors. some of these genes provide new hypotheses for elucidating the mechanisms of cobalt intracellular chemical toxicity. targeted biological approaches might confirm their biochemical role in the cobalt response. regarding biomarkers, we have highlighted the down-regulation of timp a gene coding for a secreted protein. timp modulation was confirmed at protein level, in a dose-dependent manner, in the supernatant of exposed a cells. timp provides a putative biomarker of cobalt toxicity that will be further studied on animal models. 
occupational exposure to co occurs mainly via inhalation and leads to various lung diseases. the modulated genes include potential cobalt carriers, tumor suppressors or transcription factors and genes linked to the stress response. of those, timp was found to be down-regulated and this modulation was confirmed, in a dose-dependent manner, at protein level in the supernatant of exposed cells. a putative biomarker of cobalt toxicity was identified. the purpose of this work was to identify potential signatures of acute cobalt exposure using a toxicogenomic approach. a time course transcriptome analysis was performed on a human pulmonary cells, leading to the identification of genes which are repressed or induced in response to soluble co. a group of of these genes, representing the main biological functions, was assessed by quantitative rt-pcr. the expression profiles of six of them were then tested by quantitative rt-pcr in a time-dependent manner and three modulations were confirmed by western blotting. data analysis focused on some cellular processes and protein targets that are thought to be relevant for carcinogenesis, transport and biomarker research. we also identified nine genes coding for secreted proteins as candidates for biomarker research.
 it has been estimated that more than million workers in the united states are exposed to cobalt. occupational exposure to co occurs mainly via inhalation and leads to various lung diseases. cobalt is classified by the iarc as a possible human carcinogen. although there is evidence for in vivo and in vitro toxicity, the mechanisms of cobalt-induced lung toxicity are not fully known. the purpose of this work was to identify potential signatures of acute cobalt exposure using a toxicogenomic approach. data analysis focused on some cellular processes and protein targets that are thought to be relevant for carcinogenesis, transport and biomarker research.  a time course transcriptome analysis was performed on a human pulmonary cells, leading to the identification of genes which are repressed or induced in response to soluble co. a group of of these genes, representing the main biological functions, was assessed by quantitative rt-pcr. the expression profiles of six of them were then tested by quantitative rt-pcr in a time-dependent manner and three modulations were confirmed by western blotting. the modulated genes include potential cobalt carriers, tumor suppressors or transcription factors and genes linked to the stress response. we also identified nine genes coding for secreted proteins as candidates for biomarker research. of those, timp was found to be down-regulated and this modulation was confirmed, in a dose-dependent manner, at protein level in the supernatant of exposed cells.  most of these genes have never been described as related to cobalt stress and provide original hypotheses for further study of the effects of this metal ion on human lung epithelial cells. a putative biomarker of cobalt toxicity was identified. 