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Draft Monograph on Cobalt and Certain Cobalt Compounds
Stan Atwood, MS, DABT Integrated Laboratory Systems, Inc.
Contractor supporting the ORoC
National Institute of Environmental Health Sciences July 22, 2015
Mechanistic and Other Relevant Data
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Outline
Mechanistic and Other Relevant Data
Disposition and Toxicokinetics Mechanistic and Other Relevant Data – Cobalt toxicity: particles versus ions – Proposed modes of action – Cobalt and certain cobalt compounds as a class
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Diet and inhalation are important exposure pathways Cobalt Disposition and Toxicokinetics
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymphatic System
Lymph Nodes
Interstitium
Arterial B
lood
Venous Blood
Mucociliary Clearance
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Absorption from the GI tract is highly variable (5% to > 90%)
Dietary Exposure
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymphatic System
Lymph Nodes
Interstitium
Arterial B
lood
• Aqueous (20% to 45%)
• Solid (10% to 25%)
• Women 2X > men
• Water soluble > insoluble Venous B
lood
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Soluble compounds/particles are rapidly absorbed Inhalation Exposure
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymphatic System
Lymph Nodes
Interstitium
Arterial B
lood
Venous Blood
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Large particles cleared by mucociliary action Inhalation Exposure
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymphatic System
Lymph Nodes
Interstitium
Arterial B
lood
Mucociliary Clearance
Venous Blood
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Smaller particles reach the lower airways Inhalation Exposure
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymph Nodes
Interstitium Arterial B
lood
– May dissolve and are rapidly absorbed – Phagocytized by alveolar macrophages – Enter lung cells via endocytosis – Nanoparticles may translocate directly to blood and lymph
Venous Blood Lymphatic System
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Absorbed cobalt rapidly distributes to all tissues Cobalt Disposition
Diet
GI Tract
Feces
Liver
Urine
Kidney
Heart and other tissues
Respiratory Tract
Airborne Particles
Lymph Nodes
Interstitium
Arterial B
lood
Venous Blood
• Highest in liver, kidney, heart, spleen – Stored body Co does not significantly accumulate with age
• Free fraction in blood (5% to 12%) • Insoluble particles/compounds may be retained in the lungs
Lymphatic System
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Much of absorbed cobalt is excreted within the first week
Cobalt Excretion
Diet
GI Tract
Feces
Liver
Urine
Kidney
Other Organs and Tissues
Respiratory Tract
Airborne Particles
Lymphatic System
Lymph Nodes
Interstitium
Arterial B
lood
Venous Blood
• Bile feces (2% to 12%) • Urine (30% to 60%) • Multiphasic elimination
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Clarifications?
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– Comment on whether the information on disposition and toxicokinetics is clear, technically correct, and objectively presented.
– Identify any information that should be added or deleted.
Reviewer Comments
Cobalt Disposition and Toxicokinetics
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Outline
Mechanistic and Other Relevant Data
Disposition and Toxicokinetics Mechanistic and Other Relevant Data – Cobalt toxicity: particles versus ions – Proposed modes of action – Cobalt and certain cobalt compounds as a class
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Cobalt and cobalt compounds affect similar pathways associated with carcinogenesis
Potential Modes of Action
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• Cytotoxicity/Inflammation – Nanoparticles > microparticles – Relatively soluble particles > ions > poorly soluble particles
• ROS – Nano- and microparticles > ions
– Nanoparticles activate neutrophils: ROS/proinflammatory cytokines
• For particles, direct cell contact was necessary to induce effects
• Co2+ ions responsible for effects, differences partially explained by cellular uptake mechanisms
Cobalt particles and ions have similar biological effects in vitro and in vivo
Mechanistic and Other Relevant Data
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Poorly soluble cobalt particles dissolve in the lysosomes
Cellular Uptake of Cobalt Ions and Particles
Co3O4 Particles (IC25 = 50 μg/ml)
CoCl2 (IC25 = 2.9 μg/ml)
Co2+
Co2+
Co2+
Co2+
Extracellular solubilized cobalt (0.11 μg/ml)
Intracellular solubilized cobalt (6.7 fg/cell)
Intracellular particulate cobalt (9,500 fg/cell)
Intracellular solubilized cobalt (5.4 fg/cell) lysosome
nucleus
Ion channel
Co2+
Co2+
endocytosis
Co2+
Co2+
Co2+ Co2+
NOT TOXIC
Source: Ortega et al. 2014
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Cobalt and cobalt compounds affect similar pathways associated with carcinogenesis
Potential Modes of Action
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• In vitro studies generally consistent for all forms tested
– Bacteria: mostly negative
– Human and rodent cells • Clastogenic (CA, MN, SCE, aneuploidy) • DNA damage/strand breaks • Mutations (mixed results) • Cell transformation
• In vivo (few studies)
– Mixed: Some evidence DNA/chromosome damage in rodents
– Humans (inadequate data)
Cobalt and cobalt compounds induced a similar spectrum of genotoxic and related effects
Genotoxic and Related Effects
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• Inhibits nucleotide excision repair (NER)
– Inhibits incision and polymerization steps of NER
– Increased DNA damage from UV exposure
• Substitute for Zn(II) in zinc finger domains of DNA-repair proteins and transcription factors
– p53 (repair of oxidative damage)
– Xeroderma pigmentosum group A (XPA)
– Poly(ADP-ribose)polymerase (PARP)
• Competition with Mg(II) for binding to DNA polymerases
Cobalt inhibits DNA repair
Genotoxic and Related Effects
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(1) Co(0) + O2 Co(I) + O2
(2) Co(I)−OO Co(I) + H2O2
(3) Co(I) + H2O2 Co(II) + OH + OH−
(4) [Co(II)−chelate] + H2O2 Co(II) + OH + OH−
Sources: Lee et al. 2012, Jomova and Valko 2011, Leonard et al. 1998
Cobalt is a redox-active metal
Oxidative Stress
Co(I)−OO SOD
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• NF-κB – Activation is linked to cancer
– Binds DNA: regulates cell growth & survival, differentiation, cytokine production, inflammation, angiogenesis
• Activator protein-1 (AP-1) – Participates in oncogenic transformation
– Regulates cell proliferation, apoptosis
• Promotes tumor growth by dysregulation of cell growth, proliferation, and survival
ROS activate redox transcription factors
Oxidative Stress
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• Cobalt salts (inorganic and organic) strongly active in Nrf2/ARE assay (Tox21)
• Increased sensitivity of 8-oxoguanine-DNA glycosylase (Ogg) knockout mouse embryo fibroblasts
• Cobalt particles induced dose-dependent increase in ROS in human and animal cells in vitro
• Evidence of oxidative damage in vivo in rodents (lung, liver, kidney)
• Lung tumors in rodents induced by cobalt sulfate and cobalt metal had increased frequency of G to T transversion mutations in K-ras
Cobalt and cobalt compounds cause oxidative stress/damage
ROS and Oxidative Damage
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• Cobalt well known to mimic hypoxia in vitro and in vivo
– Soluble cobalt salts (CoCl2 and CoSO4)
– Cobalt metal nanoparticles
– Poorly soluble cobalt oxide (Co3O4)
• Possible mechanism(s)
– Cobalt replaces iron in regulatory oxygenases, prevents hydroxylation of HIF and subsequent ubiquitination and degradation
– Depletes intracellular ascorbate, deactivates prolyl hydrolase activity, prevents hydroxylation of HIF
Cobalt and cobalt compounds stabilize HIF-1α
Hypoxia Inducible Factor-1α
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• Regulates > 100 hypoxia-responsive genes – VEGF, other angiogenic growth factors
– Erythrocytosis
– Inflammatory factors
– Cell proliferation
– Apoptosis
• Major role in adaption of cancer cells to hypoxia
• Overexpression/stabilization in more that 70% of human cancers and associated with poor clinical outcome.
Cellular effects of HIF-1α stabilization
Hypoxia Inducible Factor-1α
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• Toxicity for all cobalt forms tested attributed primarily to the cobalt ion
• Cobalt metal and water soluble and poorly water soluble cobalt compounds release cobalt ions in biological fluids and have similar biological effects
Rationale for evaluating cobalt and certain cobalt compounds as a class
Mechanistic and Other Relevant Data
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Similar properties and biological effects
Cobalt and Cobalt Compounds as a Class
* Dissolution of cobalt particles in lysosomal fluid is a key component for the proposed mechanisms
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• Carcinogenic to lung and adrenal gland
• K-ras mutation spectra in lung tumors
• Spectrum of inflammatory lesions in lung and extrapulmonary effects in rats
• Similar clinical findings (erythrocytosis)
In vivo studies of cobalt sulfate and cobalt metal support common underlying mechanisms
Cobalt and Cobalt Compounds as a Class
Source: Behl et al. 2015, NTP 2014, 1998
Cobalt sulfate Cobalt metal
© User:Adam Rędzikowski / Wikimedia Commons / CC-BY-SA-4.0 Int.
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* Cobalt metal
– Pancreatic islet tumors (exposure related)
– Mononuclear cell leukemia (exposure related)
– Kidney tumors (equivocal)
Cobalt and cobalt compounds induced similar carcinogenic effects in rodents
Cobalt and Cobalt Compounds as a Class
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Clarifications?
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– Comment on whether the mechanistic and other relevant data are clear, technically correct, and objectively presented.
– Comment on whether the mechanistic and other relevant data are appropriate for evaluating the biological plausibility of carcinogenic effects of cobalt and certain cobalt compounds in humans.
– Comment on whether the rationale for evaluating cobalt and certain cobalt compounds as a class is clear and scientifically sound.
– Identify any information that should be added or deleted.
Reviewer Comments
Mechanistic and Other Relevant Data