Search

Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6 +/- ). In the corneal epithelium of PAX6 +/- mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl 2 treatment., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The widespread presence and distribution of metal-based nanoparticles (NPs) in soil is threatening crop growth and food security. However, little is known about the fate of Co 3 O 4 NPs in the soil-soybean system and their phytotoxicity. The study demonstrated the effects of Co 3 O 4 NPs on soybean growth and yield in soil after 60 days and 140 days, and compared them with the phytotoxic effects of Co 2+ . The results showed that Co 3 O 4 NPs (10-500 mg/kg) had no significant toxic effect on soybeans. Soil available Co content was significantly increased under 500 mg/kg Co 3 O 4 NPs treatment. Compared with Co 2+ , Co 3 O 4 NPs mainly accumulated in roots and had limited transport to the shoots, which was related to the particle size, surface charge and chemical stability of Co 3 O 4 NPs. The significant accumulation of Co 3 O 4 NPs in roots further led to a significant decrease in root antioxidant enzyme activity and changes in functional gene expression. Co 3 O 4 NPs reduced soybean yield after 140 days, but interestingly, at specific doses, it increased grain nutrients (Fe content increased by 17.38% at 100 mg/kg, soluble protein and vitamin E increased by 14.34% and 16.81% at 10 mg/kg). Target hazard quotient (THQ) assessment results showed that consuming soybean seeds exposed to Co 3 O 4 NPs (≥100 mg/kg) and Co 2+ (≥10 mg/kg) would pose potential health risks. Generally, Co 3 O 4 NPs could exist stably in the environment and had lower environmental risks than Co 2+ . These results help to better understand the environmental behavior and plant effect mechanisms of Co 3 O 4 NPs in soil-plant systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl 2 ), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl 2 concentrations used typically range from 100 to 600 μM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl 2 . In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cobalt nanoparticles (CoNPs) have been widely used in industry given their physical, chemical and magnetic properties; however, CoNPs may cause neurological symptoms and diseases in human, yet their mechanisms of toxicity remain unknown. Here, we used male Wistar rats to investigate differences in the toxic effects associated with CoNPs and CoCl 2 . Upon exposure to CoCl 2 , and 96 nm or 123 nm CoNPs at the same concentration, the Co 2+ content in CoCl 2 group was significantly higher than that in either the CoNPs groups in brain tissues and blood, but lower in liver. Significant neural damage was observed in both hippocampus and cortex of the temporal lobe. Increase malondialdehyde (MDA) content and CASPASE 9 protein level were associated both with CoCl 2 and CoNPs treatments, consistent with lipid perioxidation and apoptosis. Heme oxygenase-1 and (NF-E2) p45-related factor-2 protein levels were elevated in response to 96 nm CoNPs exposure. In PC12 cells, NRF2 downregulation led to reduced cell viability and increased apoptotic rate. In conclusion, both CoNPs and CoCl 2 cause adverse neural effects, with nanoparticles showing greater neurotoxic potency. In addition, NRF2 protects neural cells from damage induced by CoCl 2 and CoNPs by activating downstream antioxidant responses., (Copyright © 2019. Published by Elsevier Inc.)

The Retinal Pigment Epithelium (RPE) is a monolayer of cells located above the choroid. It mediates human visual cycle and nourishes photoreceptors. Hypoxia-induced oxidative stress to RPE is a vital cause of retinal degeneration such as the Age-related Macular Degeneration. Most of these retinal diseases are irreversible with no efficient treatment, therefore protecting RPE cells from hypoxia stress is an important way to prevent or slow down the progression of retinal degeneration. Betulinic acid (BA) and betulin (BE) are pentacyclic triterpenoids with anti-oxidative property, but little is known about their effect on RPE cells. We investigated the protective effect of BA, BE and their derivatives against cobalt chloride-induced hypoxia stress in RPE cells. Human ARPE-19 cells were exposed to BA, BE and their eighteen derivatives (named as H3H20) that we customized through replacing moieties at C3 and C28 positions. We found that cobalt chloride reduced cell viability, increased Reactive Oxygen Species (ROS) production as well as induced apoptosis and necrosis in ARPE-19 cells. Interestingly, the pretreatment of 3-O-acetyl-glycyl- 28-O-glycyl-betulinic acid effectively protected cells from acute hypoxia stress induced by cobalt chloride. Our immunoblotting results suggested that this derivative attenuated the cobalt chloride-induced activation of Akt, Erk and JNK pathways. All findings were further validated in human primary RPE cells. In summary, this BA derivate has protective effect against the acute hypoxic stress in human RPE cells and may be developed into a candidate agent effective in the prevention of prevalent retinal diseases., (Copyright © 2018. Published by Elsevier Ltd.)

Hypoxia is a key pathological process involved in many cutaneous diseases. Nuclear factor E2-related factor 2 (NRF2) is a central regulator of antioxidant response element (ARE)-dependent transcription and plays a pivotal role in the cellular adaptive response to oxidative stress. Kelch-like ECH-associated protein 1 (KEAP1) is a cullin-3-adapter protein that represses the activity of NRF2 by mediating its ubiquitination and degradation. In the present study, we examined the role of NRF2 signaling pathway in the cytotoxicity induced by cobalt chloride(CoCl 2 ), a hypoxia-mimicking agent, in human keratinocyte HaCaT cells with stable knockdown of NRF2 (NRF2-KD) and KEAP1 (KEAP1-KD). Acute CoCl 2 exposure markedly increased the levels of intracellular reactive oxygen species (ROS), and resulted in hypoxic damage and cytotoxicity of HaCaT cells. Stable knockdown of NRF2 dramatically reduced the expression of many antioxidant enzymes and sensitized the cells to acute CoCl 2 -induced oxidative stress and cytotoxicity. In contrast, KEAP1-KD cells observably enhanced the activity of NRF2 and ARE-regulated genes and led to a significant resistance to CoCl 2 -induced cellular damage. In addition, pretreatment of HaCaT cells with tert-butylhydroquinone, a well-known NRF2 activator, protected HaCaT cells from CoCl 2 -induced cellular injury in a NRF2-dependent fashion. Likewise, physical hypoxia-induced cytotoxicity could be significantly ameliorated through NRF2 signaling pathway in HaCaT cells. Together, our results suggest that NRF2 signaling pathway is involved in antioxidant response triggered by CoCl 2 -induced oxidative stress and could protect human keratinocytes against acute CoCl 2 -induced hypoxic cytotoxicity., (Copyright © 2018. Published by Elsevier Inc.)

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl 2 . While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl 2 induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl 2 treatment. The CoCl 2 -dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl 2 -induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl 2 -treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p<0.05) amounts of Co and Cr ions into the culture medium, and significant (p<0.05) cellular uptake of both ions. There was also an increase (p<0.05) in apoptosis after a 48h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p<0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions+debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tungsten carbide cobalt (WC-Co) has been recognized as a workplace inhalation hazard in the manufacturing, mining and drilling industries by the National Institute of Occupational Safety and Health. Exposure to WC-Co is known to cause "hard metal lung disease" but the relationship between exposure, toxicity and development of disease remain poorly understood. To better understand this relationship, the present study examined the role of WC-Co particle size and internalization on toxicity using lung epithelial cells. We demonstrated that nano- and micro-WC-Co particles exerted toxicity in a dose- and time-dependent manner and that nano-WC-Co particles caused significantly greater toxicity at lower concentrations and shorter exposure times compared to micro-WC-Co particles. WC-Co particles in the nano-size range (not micron-sized) were internalized by lung epithelial cells, which suggested that internalization may play a key role in the enhanced toxicity of nano-WC-Co particles over micro-WC-Co particles. Further exploration of the internalization process indicated that there may be multiple mechanisms involved in WC-Co internalization such as actin and microtubule based cytoskeletal rearrangements. These findings support our hypothesis that WC-Co particle internalization contributes to cellular toxicity and suggest that therapeutic treatments inhibiting particle internalization may serve as prophylactic approaches for those at risk of WC-Co particle exposure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt-chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl2 doses (from 5 to 200μM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl2 doses for prolonged time points. Furthermore, CoCl2 treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical `pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tungsten alloys are composed of tungsten microparticles embedded in a solid matrix of transition metals such as nickel, cobalt, or iron. To understand the toxicology of these alloys, male F344 rats were intramuscularly implanted with pellets of tungsten/nickel/cobalt, tungsten/nickel/iron, or pure tungsten, with tantalum pellets as a negative control. Between 6 and 12 months, aggressive rhabdomyosarcomas formed around tungsten/nickel/cobalt pellets, while those of tungsten/nickel/iron or pure tungsten did not cause cancers. Electron microscopy showed a progressive corrosion of the matrix phase of tungsten/nickel/cobalt pellets over 6 months, accompanied by high urinary concentrations of nickel and cobalt. In contrast, non-carcinogenic tungsten/nickel/iron pellets were minimally corroded and urinary metals were low; these pellets having developed a surface oxide layer in vivo that may have restricted the mobilization of carcinogenic nickel. Microarray analysis of tumors revealed large changes in gene expression compared with normal muscle, with biological processes involving the cell cycle significantly up-regulated and those involved with muscle development and differentiation significantly down-regulated. Top KEGG pathways disrupted were adherens junction, p53 signaling, and the cell cycle. Chromosomal enrichment analysis of genes showed a highly significant impact at cytoband 7q22 (chromosome 7) which included mouse double minute (MDM2) and cyclin-dependant kinase (CDK4) as well as other genes associated with human sarcomas. In conclusion, the tumorigenic potential of implanted tungsten alloys is related to mobilization of carcinogenic metals nickel and cobalt from corroding pellets, while gene expression changes in the consequent tumors are similar to radiation induced animal sarcomas as well as sporadic human sarcomas., (Published by Elsevier Inc.)

Significant controversy over the environmental and public health impact of depleted uranium use in the Gulf War and the war in the Balkans has prompted the investigation and use of other materials including heavy metal tungsten alloys (HMTAs) as nontoxic alternatives. Interest in the health effects of HMTAs has peaked since the recent discovery that rats intramuscularly implanted with pellets containing 91.1% tungsten/6% nickel/2.9% cobalt rapidly developed aggressive metastatic tumors at the implantation site. Very little is known, however, regarding the cellular and molecular mechanisms associated with the effects of inhalation exposure to HMTAs despite the recognized risk of this route of exposure to military personnel. In the current study military-relevant metal powder mixtures consisting of 92% tungsten/5% nickel/3% cobalt (WNiCo) and 92% tungsten/5% nickel/3% iron (WNiFe), pure metals, or vehicle (saline) were instilled intratracheally in rats. Pulmonary toxicity was assessed by cytologic analysis, lactate dehydrogenase activity, albumin content, and inflammatory cytokine levels in bronchoalveolar lavage fluid 24h after instillation. The expression of 84 stress and toxicity-related genes was profiled in lung tissue and bronchoalveolar lavage cells using real-time quantitative PCR arrays, and in vitro assays were performed to measure the oxidative burst response and phagocytosis by lung macrophages. Results from this study determined that exposure to WNiCo and WNiFe induces pulmonary inflammation and altered expression of genes associated with oxidative and metabolic stress and toxicity. Inhalation exposure to both HMTAs likely causes lung injury by inducing macrophage activation, neutrophilia, and the generation of toxic oxygen radicals., (Published by Elsevier Inc.)

Nickel and cobalt are heavy metals found in land, water, and air that can enter the body primarily through the respiratory tract and accumulate to toxic levels. Nickel compounds are known to be carcinogenic to humans and animals, while cobalt compounds produce tumors in animals and are probably carcinogenic to humans. People working in industrial and manufacturing settings have an increased risk of exposure to these metals. The cytotoxicity of nickel and cobalt has individually been demonstrated; however, the underlying mechanisms of co-exposure to these heavy metals have not been explored. In this study, we investigated the effect of exposure of H460 human lung epithelial cells to nickel and cobalt, both alone and in combination, on cell survival, apoptotic mechanisms, and the generation of reactive oxygen species and double strand breaks. For simultaneous exposure, cells were exposed to a constant dose of 150 μM cobalt or nickel, which was found to be relatively nontoxic in single exposure experiments. We demonstrated that cells exposed simultaneously to cobalt and nickel exhibit a dose-dependent decrease in survival compared to the cells exposed to a single metal. The decrease in survival was the result of enhanced caspase 3 and 7 activation and cleavage of poly (ADP-ribose) polymerase. Co-exposure increased the production of ROS and the formation of double strand breaks. Pretreatment with N-acetyl cysteine alleviated the toxic responses. Collectively, this study demonstrates that co-exposure to cobalt and nickel is significantly more toxic than single exposure and that toxicity is related to the formation of ROS and DSB., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Metals and metalloid species are involved in homeostasis in energy systems such as glucose metabolism. Enlarged adipocytes are one of the most important causes of obesity-associated diseases. In this study, we studied the possibility that various metals, namely, CoCl(2), HgCl(2), NaAsO(2) and MnCl(2) pose risk to or have beneficial effects on white adipose tissue (WAT). Exposure to the four metals resulted in decreases in WAT weight and the size of enlarged adipocytes in mice fed a high-fat diet (HFD) without changes in liver weight, suggesting that the size and function of adipocytes are sensitive to metals. Repeated administration of CoCl(2) significantly increased serum leptin, adiponectin and high-density lipoprotein (HDL) cholesterol levels and normalized glucose level and adipose cell size in mice fed HFD. In contrast, HgCl(2) treatment significantly decreased serum leptin level with the down-regulation of leptin mRNA expression in WAT and a reduction in adipocyte size. Next, we tried to investigate possible factors that affect adipocyte size. Repeated exposure to HgCl(2) significantly decreased the expression levels of factors upon the regulation of energy such as the PPARα and PPARγ mRNA expression levels in adipocytes, whereas CoCl(2) had little effect on those genes expressions compared with that in the case of the mice fed HFD with a vehicle. In addition, repeated administration of CoCl(2) enhanced AMPK activation in a dose-dependent manner in the liver, skeletal muscle and WAT; HgCl(2) treatment also enhanced AMPK activation in the liver. Thus, both Co and Hg reduced WAT weight and the size of enlarged adipocytes, possibly mediated by AMKP activation in the mice fed HFD. However, inorganic cobalt may have a preventive role in obesity-related diseases through increased leptin, adiponectin and HDL-cholesterol levels, whereas inorganic mercury may accelerate the development of such diseases. These results may lead to the development of new approaches to establishing the role of metals in adipose tissue of obesity-related diseases., (Copyright © 2011. Published by Elsevier Inc.)

Tungsten alloys (WA) have been introduced in an attempt to find safer alternatives to depleted uranium and lead munitions. However, it is known that at least one alloy, 91% tungsten-6% nickel-3% cobalt (WNC-91-6-3), causes rhabdomyosarcomas when fragments are implanted in rat muscle. This raises concerns that shrapnel, if not surgically removable, may result in similar tumours in humans. There is therefore a clear need to develop rapid and robust in vitro methods to characterise the toxicity of different WAs in order to identify those that are most likely to be harmful to human health and to guide development of new materials in the future. In the current study we have developed a rapid visual in vitro assay to detect toxicity mediated by individual WA particles in cultured L6-C11 rat muscle cells. Using a variety of techniques (histology, comet assay, caspase-3 activity, oxidation of 2'7'-dichlorofluorescin to measure the production of reactive oxygen species and whole-genome microarrays) we show that, in agreement with the in vivo rat carcinogenicity studies, WNC-91-6-3 was the most toxic of the alloys tested. On dissolution, it produces large amounts of reactive oxygen species, causes significant amounts of DNA damage, inhibits caspase-3, triggers a severe hypoxic response and kills the cells in the immediate vicinity of the alloy particles within 24h. By combining these in vitro data we offer a mechanistic explanation of the effect of this alloy in vivo and show that in vitro tests are a viable alternative for assessing new alloys in the future., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5-15%). WC-Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC-Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC-Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-kappaB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P(+)). Our results demonstrated that nano-WC-Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P(+) cell growth/proliferation than observed after exposure of cells to fine WC-Co. In addition, nano-WC-Co activated AP-1 and NF-kappaB more efficiently in JB6(+/+) cells as compared to fine WC-Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC-Co. Nano- and fine-sized WC-Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC-Co. Finally, co-incubation of the JB6(+/+) cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC-Co-induced toxicity and AP-1 activation.

Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO(2) to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO(2) and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO(2), at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression(..) Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO(2). Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells after exposure to Nano-Co, but not to Nano-TiO(2). However, neither Nano-Co nor Nano-TiO(2) exposure led to any transcriptional change of TIMP-1. The decrease of TIMP-2 after exposure to Nano-Co was also inhibited by pre-treatment with ROS scavengers or inhibitors. Our results also showed that pre-treatment of U937 cells with AP-1 inhibitor, curcumin, or the PTK specific inhibitor, herbimycin A or genistein, prior to exposure to Nano-Co, significantly abolished Nano-Co-induced pro-MMP-2 and-9 activity. Our results suggest that Nano-Co causes an imbalance between the expression and activity of MMPs and their inhibitors which is mediated by the AP-1 and tyrosine kinase pathways due to oxidative stress.

This study reports the efficacy of cobalt preconditioning in preventing hypobaric hypoxia induced vascular leakage (an indicator of cerebral edema) using male Sprague-Dawley rats as model system. Exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, reactive oxygen species (ROS), nitric oxide (NO), and vascular endothelial growth factor (VEGF) levels. There was a marked increase in Nuclear Factor kappaB (NFkappaB) DNA binding activity and levels of pro-inflammatory cytokines such as Monocyte chemoattractant protein (MCP-1), Interferon-gamma (IFN-gamma), Interleukin-1 (IL-1), and Tumor Necrosis Factor-alpha (TNF-alpha) and cell adhesion molecules such as Vascular Cell Adhesion Molecule-1 (VCAM-1), and P-selectin. Chemical preconditioning by cobalt for 7 days (12.5 mg Co/kg b.w., oral) significantly attenuated cerebral vascular leakage and the expression of inflammatory mediators induced by hypoxia. Administration of NFkappaB inhibitor, curcumin (50 mg/kg b.w.; i.p.) appreciably inhibited hypoxia induced vascular leakage indicating the involvement of NFkappaB in causing vascular leakage. Interestingly, cobalt when administered at 12.5 mg Co/kg b.w. (i.p.), 1 h before hypoxia could not prevent the vascular leakage indicating that cobalt per se did not have an effect on NFkappaB. The lower levels of NFkappaB observed in the brains of cobalt administered animals might be due to higher levels of antioxidant and anti-inflammatory proteins (hemeoxygenase-1 and metallothionein). To conclude cobalt preconditioning inhibited hypobaric hypoxia induced cerebral vascular leakage by lowering NFkappaB DNA binding activity and its regulated pro-inflammatory mediators. This is contemplated to be mediated by cobalt induced reduction in ROS/NO and increase in HO-1 and MT.

Hard metals consist of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC-Co particle mixture was shown to be carcinogenic in exposed workers. The in vitro mutagenic/apoptogenic potential of WC-Co in human peripheral blood mononucleated cells was previously demonstrated by us. This study aimed at obtaining a broader view of the pathways responsible for WC-Co induced carcinogenicity, and in particular genotoxicity and apoptosis. We analyzed the profile of gene expression induced in vitro by WC-Co versus control (24 h treatment) in human PBMC and monocytes using microarrays. The most significantly up-regulated pathways for WC-Co treated PBMC were apoptosis and stress/defense response; the most down-regulated was immune response. For WC-Co treated monocytes the most significantly up- and down-regulated pathways were nucleosome/chromatin assembly and immune response respectively. Quantitative RT-PCR data for a selection of the most strongly modulated genes (HMOX1, HSPA1A, HSPA1L, BNIP3, BNIP3L, ADORA2B, MT3, PLA2G7, TNFAIP6), and some additionally chosen apoptosis related genes (BCL2, BAX, FAS, FASL, TNFalpha), confirmed the microarray data after WC-Co exposure and demonstrated limited differences between the Co-containing compounds. Overall, this study provides the first analysis of gene expression induced by the WC-Co mixture showing a large profile of gene modulation and giving a preliminary indication for a hypoxia mimicking environment induced by WC-Co exposure.

The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

Hypoxia inducible factor-1 (HIF-1) is the main transcriptional factor activated by hypoxia. Besides the well-described role assigned to HIF-1 in the adaptation of cells to hypoxia, different recent data describe a possible role for HIF-1 in the modulation of apoptosis. However, this precise role is not yet clearly understood. In this study, chemical and physiological hypoxia, which were shown to induce HIF-1alpha stabilization and HIF-1 activation, were shown to inhibit apoptosis induced in HepG2 cells by two different pro-apoptotic conditions, serum deprivation- and t-BHP-induced oxidative stress. Indeed, hypoxia reduced DNA fragmentation, caspase activation, and PARP cleavage induced by these two pro-apoptotic conditions. These results are very interesting because it is a clear demonstration that hypoxia and chemical hypoxia have a direct protective effect on apoptotic cell death induced by two different stimuli. This observation is an important data in understanding how tumor growth can occur in challenging environmental conditions.

Inhalation of dust from hard metal (HM), a mixture of tungsten carbide, cobalt, and other metals, can cause interstitial alveolitis, fibrosis, and asthma in the workplace. Some effects of HM could occur after the metals dissolve in the lung. We examined whether chloride salts of metals in HM alloys can elicit responses or modify reactivity to methacholine (MCh) or responses to electric field stimulation (EFS) in guinea pig tracheal strips. In unstimulated strips, Co(2+), Cd(2+), and Ni(2+) evoked contractions (>3 x 10(-6) M), while Ta(5+), Zn(2+), Cr(2+), and Cr(3+) caused weak relaxations (>10(-5) M). In strips contracted with MCh (3 x 10(-7) M), Co(2+) and Ni(2+) also caused relaxation in lower concentrations while the other metals caused weak relaxation only in high concentrations (>10(-4) M). The metals were generally without effect on reactivity to MCh, except that Cd(2+) inhibited and Ni(2+) potentiated some responses. The effects of selected metals (10(-6) M; Cr(3+), Ni(2+), Cd(2+), and Co(2+)) on EFS-induced contractile and relaxant responses were examined (+/-MCh; +/-10(-6) M indomethacin (Indo), 30 min). No metal had any effect on the excitatory nonadrenergic, noncholinergic-mediated contraction phase. Cd(2+) and Ni(2+) inhibited cholinergically mediated contractions of unstimulated strips (+Indo), whereas Cr(3+) both inhibited (-MCh, -Indo) and potentiated (-Indo,+MCh; +Indo, +MCh) contractile responses. Cr(3+) was the only metal to inhibit the inhibitory nonadrenergic, noncholinergic-mediated relaxation phase (+/-MCh; -Indo). Co(2+) had no effect at all. The results suggest that smooth muscle tone and nerves in the airways could be targets of cationic metals after they dissolve in the lung.

It has been shown that cobalt (Co) and the mixture cobalt-tungsten carbide (CoWC) are cytotoxic for alveolar macrophages (AM) and alveolar type II cells (AT-II), but the exact mechanisms of toxicity are not entirely elucidated. In this study, we exposed primary cultures of AT-II and AM, in vitro, to different forms of Co (Co particles, CoWC particles, CoCl(2)) and assessed cell damage using the dimethylthiazol diphenyl tetrazolium bromide test. In some experiments, inserts were used to separate the particles from the cells. The results show that AT-II are twice as sensitive to the effects of 100 microg Co particles/well (1.88 cm(2)) than AM. For this latter cell type, the presence of WC almost doubled (at 25 microg Co/well) the toxic effects compared to pure Co, but this synergy between Co and WC only occurred if the particles were in close contact with the cells. Lactalbumin and, to a lesser degree, EDTA were able to reduce the toxicity of Co, CoWC, and CoCl(2) for AT-II and AM. CoCl(2) showed a similar toxicity for AT-II and AM. The use of Co-conditioned medium revealed that Co particles are "aged" after having been incubated for 24 h in an aqueous medium and are then no longer able to cause the same degree of cell damage as fresh Co particles (71 versus 15% viability for 100 microg Co/well). The time course of the toxicity of the different forms of Co for AT-II and AM showed different patterns in causing cell damage, suggesting different action mechanisms. Evaluation of cell damage by electron microscopy was consistent with biochemical indices. Overall, our results indicate that the Co ion does play a role in the toxicity of both Co particles and CoWC particles., (Copyright 2000 Academic Press.)

To examine whether the development of hard metal (HM)-induced occupational asthma and interstitial lung disease involves alterations in nitric oxide (NO) pathways, we examined the effects of an industrial HM mixture on NO production, interactions between HM and lipopolysaccharide (LPS) on NO pathways, and alterations in airway reactivity to methacholine in rat lungs. HM (2.5 to 5 mg/100 g intratracheal) increased NO synthase (NOS; EC 1.14.23) activity of rat lungs at 24 h without increasing inducible NOS (iNOS) or endothelial NOS (eNOS) mRNA abundance or iNOS, eNOS, or brain NOS (bNOS) proteins. The increase in NOS activity correlated with the appearance histologically of nitrotyrosine immunofluorescence in polymorphonuclear leukocytes (PMN) and macrophages. Intraperitoneal injection of LPS (1 mg/kg) caused up-regulation of iNOS activity, mRNA, and protein at 8 h but not at 24 h. HM at 2.5 mg/100 g, but not at 5 mg/100 g, potentiated the LPS-induced increase in NOS activity, iNOS mRNA, and protein. However, HM decreased eNOS activity at 8 h and eNOS protein at 24 h. Whole body plethysmography on conscious animals revealed that HM caused basal airway obstruction and a marked hyporeactivity to inhaled methacholine by 6-8 h, which intensified over 30-32 h. HM-treatment caused protein leakage into the alveolar space, and edema, fibrin formation, and an increase in the number of inflammatory cells in the lungs and in the bronchoalveolar lavage. These results suggest that a HM-induced increase in NO production by pulmonary inflammatory cells is associated with pulmonary airflow abnormalities in rat lungs.

Macrophages (M phi) can be induced to produce nitric oxide (NO), which has been suggested to be important for macrophages to exercise various functions. We have previously reported that an environmental toxicant, lead (Pb), can significantly inhibit NO production by murine splenic M phis. Herein, eight additional metal ions, gold (Au), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), and zinc (Zn), were assessed. In addition to Pb, Hg and Cd significantly suppressed NO production by cytokine (interferon-gamma and tumor necrosis factor-alpha)-stimulated murine M phis. Au and Cu also were inhibitory, but less than Pb, Hg, and Cd. In contrast, Cr and Zn were not modulatory, and Ni and Co significantly enhanced NO production by cytokine-stimulated M phis. The enhancement by Ni and Co was inhibited by the arginine analog N-monomethylarginine. The metals showed different activating/inhibiting profiles when added to a cell-free (activated M phi lysate) NO-producing-system in which inducible NO synthase (iNOS) is already expressed. Cr, Cu, Pb, and Zn moderately suppressed iNOS, which suggests that they may directly modify enzyme or cofactor activity. Cd, Hg, Mg, Ni, or Co did not produce any significant effect on NO production by the cell-free system. Inhibition of NO production by Pb-exposed M phis was not due to decreased expression of iNOS nor limited to its modest direct inhibition of iNOS; thus, other mechanism(s) must be accountable for the efficient Pb-induced inhibition of NO production by M phi. Ni or Co did induce a substantial increase of iNOS protein. Overall, these observations provide additional insight into the means by which metals via inhibition or enhancement of NO production may be pathogenic, by suppression of defense mechanisms or induction of hypersensitivity, respectively.

Hard metal alloys (WC-Co) are made of a mixture of cobalt (Co; 6%) and tungsten carbide (WC; 94%) particles. Chronic inhalation of hard metal dust can lead to the development of a fibrosing alveolitis, the pathogenesis of which is still undefined. The present investigation was undertaken to assess the effect of Co, WC, and WC-Co particles on the release by lung phagocytes of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), fibronectin, and cystatin-c. The responses were compared with those induced by two other lung toxicants, i.e., crystalline silica (DQ12) and arsenic trioxide (As2O3). IL-1 and TNF-alpha activities produced in the presence and absence of LPS stimulation were measured with the aid of bioassays while fibronectin and cystatin-c were determined by latex immunoassays. In vitro, maximal noncytotoxic doses of As2O3, Co, WC, or WC-Co did not significantly affect the production of these mediators by rat alveolar macrophages. In contrast, DQ12 enhanced the production of TNF-alpha (with and without LPS stimulation) and IL-1 (after LPS stimulation) and decreased cystatin-c release (in the absence of LPS). Following a single intratracheal instillation of the different test preparations in the rat, the response of the lung phagocytes obtained by bronchoalveolar lavage (BAL) 24 hr later was examined. We were unable to detect any consistent effect of Co (0.06 mg/100 g body wt), WC (1 mg/100 g body wt), or WC-Co treatment (1 mg/100 g body wt) on the production of the above mediators. In contrast, after LPS stimulation, As2O3 (0.5 mg/100 g body wt) and DQ12 (1 mg/100 g body wt) stimulated the production of TNF-alpha and IL-1. In the absence of LPS, As2O3 stimulated fibronectin and cystatin-c production and DQ12 stimulated cystatin-c release. Since the dose of WC-Co used in vivo (1 mg/100 g body wt) caused pronounced lung inflammation (increased LDH, protein, and albumin levels in BAL fluid), we conclude that the acute lung toxicity of WC-Co particles is not mediated through enhanced production of the examined mediators by lung phagocytes.

Kainate-activated Co2+ uptake, a histochemical method that identifies cells bearing Ca2+-permeable AMPA/kainate receptors, labels approximately 15% of murine cortical neurones in cell culture. While exposure times exceeding several hours were needed for AMPA or kainate to destroy most cultured cortical neurones, the subpopulation exhibiting kainate-activated Co2+ uptake was selectively destroyed after AMPA or kainate exposures of only 10-60 min. No selective loss was seen after exposure to NMDA. Kainate toxicity on Co2+ uptake-positive neurones was dependent on extracellular Ca2+ concentration, and associated with an increase in intracellular free Ca2+ even in the absence of extracellular Na+. These results suggest that a distinct subpopulation of cortical neurones expresses AMPA/kainate receptors linked to Ca2+-permeable channels, and that this characteristic conveys enhanced vulnerability to kainate-induced, Ca2+-mediated, damage.

Genetic and related effects of cobalt compounds are reviewed and discussed with respect to mechanisms. In prokaryotic assays, Co(II) salts generally are nonmutagenic. In Saccharomyces cerevisiae, CoCl2 is mutagenic to mitochondrial genes and weakly mutagenic or nonmutagenic to chromosomal genes. In plants, Co(II) salts induced gene mutations and chromosomal aberrations. In mammalian cells in vitro, Co(II) compounds caused DNA strand breaks, sister-chromatid exchanges and aneuploidy, but not chromosomal aberrations. In two cell lines, CoCl2 was weakly mutagenic. Interestingly, the poorly soluble compound CoS caused DNA strand breaks and morphological transformation of mammalian cell lines. In contrast to its weak clastogenic and mutagenic properties, cobalt(II) exerts pronounced antimutagenicity in bacteria and mostly comutagenic effects in mammalian cells. In Escherichia coli CoCl2 lowered the frequency of mutations induced by MNNG, uv or X rays. In Chinese hamster V79 cells, CoCl2 enhanced the mutagenicity and clastogenicity of uv light but not of gamma rays. Regarding direct genotoxic mechanisms, Co(II) induces the formation of reactive oxygen species when combined with hydrogen peroxide in cell-free systems. At high (i.e., millimolar) concentrations, Co(II) also decreases the fidelity of DNA synthesis. Regarding anti- and co-mutagenic mechanisms, evidence for the interference of Co(II) with DNA repair processes is discussed. These mechanisms are regarded as relevant for the risk assessment of human exposure to cobalt in combination with other agents.

The effects of the surfactant stimulating drug ambroxol were studied in rats given paraquat (PQ, 15 mg/kg, s.c.), intratracheal cobalt chloride (CoCl2, 2.5 or 1.25 mg Co/kg) or exposed for 2 days to oxygen (greater than 95% O2). Ambroxol (50 mg/kg, i.p., twice daily) was given as a pretreatment for 4 days, or up to 7 days following pneumotoxic administration. Besides body weight evolution and lethality, various pulmonary indices were measured 2 days (O2) or 3 days (PQ and CoCl2) and 7 days after pneumotoxic treatment: wet and dry lung weight, total cell count, distribution of inflammatory cells and lactate dehydrogenase in bronchoalveolar lavage fluid, and histological damage assessed by a semiquantitative injury score. Neither form of ambroxol treatment prevented the toxicity of any of the pneumotoxic agents. The use of a combination of different indices of lung damage showed that different pneumotoxic agents may cause quite distinctive patterns of injury.

Groups of 10 F344/N rats and B6C3F1 mice of each sex were exposed to cobalt sulfate heptahydrate aerosols of 0, 0.3, 1.0, 3.0, 10, or 30 mg/m3, 6 hr per day, 5 days per week, for 13 weeks. All rats and female mice and all but 2/10 male mice exposed at the top concentration survived to the end of the studies. Polycythemia was observed in exposed rats but not in mice. Sperm motility was decreased in mice exposed at 3 mg/m3 (the lowest concentration evaluated) and at higher concentrations, and increased numbers of abnormal sperm and decreased testis and epididymal weights occurred in mice exposed to 30 mg/m3. Cobalt content in the urine of rats increased with increasing atmospheric cobalt exposure. Primary histopathologic effects were limited to the respiratory tract. Lesions in rats and mice included degeneration of the olfactory epithelium, squamous metaplasia of the respiratory epithelium, and inflammation in the nose; inflammation, necrosis, squamous metaplasia, ulcers (rats), and inflammatory polyps (rats) of the larynx; metaplasia of the trachea (mice); and fibrosis, histiocytic infiltrates, bronchiolar epithelial regeneration, and epithelial hyperplasia in the alveoli of the lung. The most sensitive tissue was the larynx, with squamous metaplasia observed in rats and mice at the lowest exposure concentration of 0.3 mg/m3. Thus, a no-observed-adverse-effect level was not reached in these studies.