Carbon Black Exposure Research Articles

N6-methyladenosine-dependent primary microRNA-126 processing activated PI3K-AKT-mTOR pathway drove the development of pulmonary fibrosis induced by nanoscale carbon black particles in rats

The pulmonary fibrosis could be caused by long-term inhalation of carbon black (CB) particles. Studies on the mechanisms of pulmonary fibrosis induced by CB are required to develop the stratagem of prevention and treatment on fibrosis. The RNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8)-dependent pri-miRNAs processing is regulated by N6-methyladenosine (m6A) modification, which targets the downstream signal pathway. However, its role in pulmonary fibrosis has not been known clearly. In the present study, rats inhaled CB at dose of 0, 5 or 30 mg/m3 for 28 days, 6 h/day, respectively. The rats inhaled CB at dose of 0 or 30 mg/m3 for 14 days, 28 days and 90 days, respectively. In vitro experiments, the normal human bronchial epithelial cell line (16HBE) was treated with CB (0, 50, 100 and 200 μg/mL) for 24 h. In vitro and vivo study, the levels of fibrosis indicators including α-SMA, vimentin, collagen-I and hydroxyproline in CB treatment groups statistically increased in dose- or time- dependent manners compared with the control. After CB treatment, PI3K-AKT-mTOR pathway was activated and regulated by miRNA-126. We found that both of m6A modifications of pri-miRNA-126 and its binding with DGCR8 were decreased after CB treatment, which resulted in the reduction of mature miRNA-126 accompanied by accumulation of unprocessed pri-miRNA-126. This work demonstrated that m6A modification of pri-miRNA-126 and its binding with DGCR8 decreases blocked miRNA-126 maturation, and then activated the PI3K/AKT/mTOR pathway, which drove the fibro genesis in the lung after CB exposure.

Microglial activation and inflammation caused by traffic-related particulate matter

Neurotoxicity caused by particulate matter (PM) has been highlighted as being a potential risk factor for neurodegenerative diseases. However, the effects of brain inflammation in response to traffic-related PM remain unclear. The objective of this study was to investigate the effects of traffic-related PM on microglial responses. We determined the cytotoxicity, oxidative stress, lipid peroxidation, inflammation, activation, autophagy, and apoptosis due to exposure to carbon black (CB) and diesel exhaust particles (DEPs) in Bv2 microglial cells. Additionally, cells were pretreated with corticosteroid to determine alterations in microglial activation and inflammation. For in vivo confirmation, Sprague Dawley (SD) rats were whole-body exposed to traffic-related PM1 (PM with an aerodynamic diameter of <1 μm) for 3 and 6 months. We observed that a decrease in cell viability and increases in dichlorodihydrofluorescein (DCFH), lactate dehydrogenase (LDH), and thiobarbituric acid-reactive substances (TBARSs) occurred due to CB and DEP. Production of interleukin (IL)-6 and soluble tumor necrosis factor (TNF)-α was significantly stimulated by CB and DEP, whereas production of cellular TNF-α was significantly stimulated by CB. Iba1 and prostaglandin E2 (PGE2) significantly increased due to CB and DEP. Consistently, we observed significant increases in Iba1 in the hippocampus of rats after 3 and 6 months of exposure to traffic-related PM1. We found that the light chain 3II (LC3II)/LC3I ratio and caspase-3 activity increased due to CB and DEP exposure. Subsequently, LDH, TBARS, LC3II/I, and caspase-3 activities did not clearly respond to corticosteroid pretreatment followed by DEP exposure in BV2 cells. Results of the present study suggested that traffic-related PM induced cytotoxicity, lipid peroxidation, microglial activation, and inflammation as well as autophagy and caspase-3 regulation in microglia. We demonstrated that microglial activation and inflammation may play important roles in the response of the brain to traffic-related PM.

Carbon black-induced detrimental effect on osteoblasts at low concentrations: Remarkably compromised differentiation without significant cytotoxicity

Due to similar aerodynamic and micro-nano sized properties between airborne particles and synthetic nanoparticles, a large number of studies have been conducted using carbon-based particles, such as carbon black (CB), carbon nanotubes and graphite, in order to achieve deeper understandings of their adverse effects on human health. It has been reported that particulate matters can aggravate morbidity of patients suffering from bone and joint diseases, e.g. arthritis. However, the molecular mechanism is still elusive thus far. Under this context, we employed two cell lines of osteoblasts, MC3T3-E1 and MG-63, upon exposure to 4 different CB samples with differential physicochemical properties in research of mechanistic insights. Our results indicated that the carbon/oxygen ratio differed in these 4 CB materials showing the order: SB4A < Printex U < C1864 < C824455. In stark contrast, their cytotoxicity and capacity to trigger reactive oxygen species (ROS) in MC3T3-E1 and MG-63 cells closely correlated to oxygen content, revealing the reverse order: SB4A < Printex U < C1864 < C824455. It would be reasonable to speculate that ROS production was a predominant cause of CB cytotoxicity, which strongly relied on the oxygen content of CB. Our study further manifested that all CB samples even at low concentrations significantly inhibited osteoblast differentiation, as reflected by remarkably reduced activity of alkaline phosphatase (ALP) and compromised expression of the differentiation-related genes. And the inhibition on osteoblast differentiation also closely correlated to oxygen content of CB samples. Taken together, our combined data recognized oxygen-associated toxicity towards osteoblasts for CBs. More importantly, we uncovered a new adverse effect of CB exposure: suppression on osteoblast differentiation, which has been overlooked in the past.

Pulmonary toxicities from a 90-day chronic inhalation study with carbon black nanoparticles in rats related to the systemical immune effects.

Background: Recent years, there occurs heavy haze pollution in northern China during wintertime. The potential influence of airborne particulate matter (PM) on human health attracts great concern. The fuel-derived PM in the inhalable size range is dominated by aggregates of nanoparticles of Carbon black (CB). However, there are still lack of evidences especially regarding long-term exposure to explain the chronic effects of nanoscaled CB and the relative mechanism.Purpose: The objective of this study was to identify the potential mechanism of chronic effects of nanoscale CB. The systemic toxicity, immune suppression or activity and local toxicity were evaluated.Methods: 32 rats were divided into 2 groups: 30 mg/m3 CB exposure (nose only, 90 d, 6h/d) and control (clean air). Half of rats were scarified after exposure and another half of rats recovered for 14 days. Eight rats in each group were executed the lung function tests using a ventilated bias flow whole body plethysmograph (WBP). SDS-PAGE protocol was used to detect the deposition and retention of CB in lung of rats. HE staining was used to observe the changes of histopathology. Cell apoptosis was examined by TUNEL assay or flow cytometry. The levels of IL-6, IL-8, IL-17 and TNF-α in serum and lung tissue were evaluated with commercially available ELISA kit. The peripheral blood cell counts were detected by Auto 5-diff hematology analyzer.Results: The lung burden of CB was 16 mg in lung of rats after a 90-day exposure by MPPD. Fourteen percentages of the amount of CB accumulated at the end of the exposure period was cleared from the lung during the 14 dys recovery period. The lung function was significantly decreased and could not recover after a short time recovery. The fibroblasts and granuloma formation were found in lung. The levels of apoptosis and DNA damages were significantly increased in lung cells after CB inhalation. The cytokines levels in lung but not in serum were significantly increased in CB exposure group. The cell counts of WBC, monocytes and neutrophils had 1.72, 3.13, and 2.73-fold increases after CB exposure, respectively. The percentages of CD4+ lymphocytes and the rates of CD4+/CD8+ were statistically increased after CB exposure. The stimulation indexes of the peripheral blood lymphocytes were significantly decreased after CB exposure. In the CB exposure group, the disrupted histomorphology of thymus and spleen were found as well as the early apoptotic thymocytes had a 2.36-fold increase.Conclusion: CB induced the localized or direct toxicity and systemic immune toxicity. The direct and systemic immune responses had a combined effect on the lung damages caused by CB.

Nanosized carbon black exposure induces neural injury: effects on nicotinamide adenine dinucleotide phosphate oxidases and endoplasmic reticulum stress

Carbon black in ambient air is believed to be the cause of many diseases; however, its potential neural toxicity and the underlying mechanisms remain poorly understood. The present study is to evaluate the toxic effects of carbon black nanoparticles, Printex 90, on the neural cell line PC-12. The study revealed that Printex 90 treatment significantly decreased cell viability, accompanied by an enormous increase in reactive oxygen species generation and a decrease in ATP. Additionally, NOX2 and NOX4, 4-hydroxynonenal, endoplasmic reticulum (ER) stress marker proteins (IRE-1α, ATF-6, GRP78, PERK and the downstream target protein CHOP) and antioxidative enzymes (glutathione and superoxide dismutase) were evaluated. It showed that Printex 90 significantly upregulated 4-hydroxynonenal, NOX2 and NOX4 expression, and the levels, or activity, of glutathione and superoxide dismutase, were markedly reduced. For the ER stress-associated proteins, Printex 90 induced a significant increase of IRE-1α, ATF-6, GRP78, p-PERK and CHOP expression. Collectively, these results demonstrate that NOX and ER stress are involved in Printex 90-mediated neural damage. Therefore, decreased ER stress and NOX-derived reactive oxygen species generation may provide compensatory protective effects and attenuate Printex 90-induced neural injury.

The Protective Effect of a Topical Mucin Secretagogue on Ocular Surface Damage Induced by Airborne Carbon Black Exposure.

Exposure to airborne particulate matter can induce ocular surface damage and inflammation. We evaluated the effects of a topical mucin secretagogue on the mitigation of ocular surface damage induced by exposure to airborne carbon black (CB). Sprague-Dawley rats were exposed to ambient CB for 2 hours twice daily for 5 days. Corneal staining score and tear lactic dehydrogenase (LDH) activity were measured to evaluate ocular surface damage. Serum immunoglobulin (Ig) G and IgE levels and the sizes of cervical lymph nodes were also measured. The expressions of interleukin (IL)-4, IL-17, and interferon (IFN)-γ were measured by Western blot analysis. Diquafosol tetrasodium was instilled six times a day for 5 days, and the extent of ocular surface damage was evaluated. After exposure to airborne CB, the median corneal staining score and LDH activity were significantly increased. Serum IgG and IgE levels and the sizes of cervical lymph nodes were also significantly increased. Additionally, the expression of IL-4 and IFN-γ was elevated in the anterior segment of the eyeball. Furthermore, the expression of IL-4, IL-17, and IFN-γ was elevated in the cervical lymph nodes. When exposed to airborne black carbon, topical diquafosol tetrasodium significantly increased tear MUC5AC concentration and decreased tear LDH activity. Exposure to airborne CB induced ocular surface damage and increased proinflammatory cytokines in the eyes and cervical lymph nodes. Topical mucin secretagogues seem to have a protective effect on the ocular surface against exposure to airborne particulate matters.

Carbon black induced DNA damage and conformational changes to mouse hepatocytes and DNA molecule: A combined study using comet assay and multi-spectra methods

Carbon black (CB), a carbonaceous nanoparticle, has been widely applied in our daily lives and used as a typical model to study environmental safety and health impacts of airborne particles. Although the potential negative effects of CB to organisms have been reported a lot, very limited work is focused on the genotoxicity of CB on molecular and cellular level simultaneously. Herein, we investigated the interaction mechanism between CB and DNA molecule in depth by multiple spectra measurement, UV–vis absorption and ionic strength measurement. The fluorescence spectroscopy, ironic strength measurement and UV absorption indicated that CB changed the structure of DNA and interacted with DNA in an electrostatic binding mode. CD (circular dichroism) spectra proved no significant effects were caused by CB on the base stacking and helicity bands of DNA, which further verified that electrostatic binding is the main binding mode between CB and DNA. On the cellular level, the comet assay shows that CB exposure could cause a remarkable DNA strand break to the mouse hepatocytes after 24 co-incubation. This combined investigation suggests that CB could cause a serious genotoxicity both on molecular and cellular level.

From the lung to the knee joint: Toxicity evaluation of carbon black nanoparticles on macrophages and chondrocytes

Carbon black (CB), a core elemental carbon component of airborne particles, has been used as a model material to study environmental safety and health impacts of airborne particles. Although potential adverse effects of CB have been reported, limited knowledge is available regarding CB-induced metabolic disorders and secondary effects distant from primary target organs, such as the effects on joints. The knee cavity is a relatively closed space along the peripheral circulation route with a slow rate of interchange of nutrition with blood. While epidemiologic studies have indicated that airborne particle exposure may affect the occurrence and severity of inflammatory knee diseases, no research has been performed to understand the potential hazardous direct/indirect interactions between particles and knee cells. Herein, we have scrutinized the toxicity of four commercial nano-sized CB samples in the lung and a distant site: knee joint. Our results indicated that CB triggered pulmonary and systemic inflammation upon inhalation exposure, and, more strikingly, CB also elicited injuries of the knee joint, as demonstrated by thickened synovial membrane, suggesting disordered cellular metabolism within the knee joint. Our data recognized the CB toxicity profiles to macrophages as characterized by pro-inflammatory reactions, and also defined an activated metabolic state of chondrocytes, as evidenced by metalloproteinase (MMP) induction. Of note, remarkable variations were also found for these changes induced by these four CB samples, due to their distinct physicochemical properties. Collectively, our results uncovered a significant toxicity of CB inhalation exposure to the knee joint, as reflected by metabolic activation of chondrocytes, and, more importantly, these findings unearthed CB-induced metabolic disorders and secondary effects owing to systemic pro-inflammatory conditions upon CB exposure, in addition to the likelihood of direct toxicity to knee cells.

Effects of physical characteristics of carbon black on metabolic regulation in mice

Potential adverse effects of human exposure to carbon black (CB) have been reported, but limited knowledge regarding CB-regulated metabolism is currently available. To evaluate how physical parameters of CB influence metabolism, we investigated CB and diesel exhaust particles (DEPs) and attempted to relate various physical parameters, including the hydrodynamic diameter, zeta potential, and particle number concentrations, to lung energy metabolism in female BALB/c mice. A body weight increase was arrested by 3 months of exposure to CB of smaller-size fractions, which was negatively correlated with pyruvate in plasma. There were no significant differences in cytotoxic lactate dehydrogenase (LDH) or total protein in bronchoalveolar lavage fluid (BALF) after 3 months of CB exposure. However, we observed alterations in acetyl CoA and the NADP/NADPH ratio in lung tissues with CB exposure. Additionally, the NADP/NADPH ratio was associated with the zeta potential of CB. Mild peribronchiovascular and interstitial inflammation and multinucleated giant cells (macrophages) with a transparent and rhomboid appearance and containing foreign bodies were observed in lung sections. We suggest that physical characteristics of CB, such as the zeta potential, may disrupt metabolism after pulmonary exposure. These results, therefore, provide the first evidence of a link between pulmonary exposure to CB and metabolism.

The Toxicological Mechanisms of Environmental Soot (Black Carbon) and Carbon Black: Focus on Oxidative Stress and Inflammatory Pathways.

The environmental soot and carbon blacks (CBs) cause many diseases in humans, but their underlying mechanisms of toxicity are still poorly understood. Both are formed after the incomplete combustion of hydrocarbons but differ in their constituents and percent carbon contents. For the first time, “Sir Percival Pott” described soot as a carcinogen, which was subsequently confirmed by many others. The existing data suggest three main types of diseases due to soot and CB exposures: cancer, respiratory diseases, and cardiovascular dysfunctions. Experimental models revealed the involvement of oxidative stress, DNA methylation, formation of DNA adducts, and Aryl hydrocarbon receptor activation as the key mechanisms of soot- and CB-induced cancers. Metals including Si, Fe, Mn, Ti, and Co in soot also contribute in the reactive oxygen species (ROS)-mediated DNA damage. Mechanistically, ROS-induced DNA damage is further enhanced by eosinophils and neutrophils via halide (Cl− and Br−) dependent DNA adducts formation. The activation of pulmonary dendritic cells, T helper type 2 cells, and mast cells is crucial mediators in the pathology of soot- or CB-induced respiratory disease. Polyunsaturated fatty acids (PUFAs) were also found to modulate T cells functions in respiratory diseases. Particularly, telomerase reverse transcriptase was found to play the critical role in soot- and CB-induced cardiovascular dysfunctions. In this review, we propose integrated mechanisms of soot- and CB-induced toxicity emphasizing the role of inflammatory mediators and oxidative stress. We also suggest use of antioxidants and PUFAs as protective strategies against soot- and CB-induced disorders.

Influence of inflammation and nitric oxide upon platelet aggregation following deposition of diesel exhaust particles in the airways.

Background and PurposeExposure to nanoparticulate pollution has been implicated in platelet‐driven thrombotic events such as myocardial infarction. Inflammation and impairment of NO bioavailability have been proposed as potential causative mechanisms. It is unclear, however, whether airways exposure to combustion‐derived nanoparticles such as diesel exhaust particles (DEP) or carbon black (CB) can augment platelet aggregation in vivo and the underlying mechanisms remain undefined. We aimed to investigate the effects of acute lung exposure to DEP and CB on platelet activation and the associated role of inflammation and endothelial‐derived NO.Experimental ApproachDEP and CB were intratracheally instilled into wild‐type (WT) and eNOS−/− mice and platelet aggregation was assessed in vivo using an established model of radio‐labelled platelet thromboembolism. The underlying mechanisms were investigated by measuring inflammatory markers, NO metabolites and light transmission aggregometry.Key ResultsPlatelet aggregation in vivo was significantly enhanced in WT and eNOS−/− mice following acute airways exposure to DEP but not CB. CB exposure, but not DEP, was associated with significant increases in pulmonary neutrophils and IL‐6 levels in the bronchoalveolar lavage fluid and plasma of WT mice. Neither DEP nor CB affected plasma nitrate/nitrite concentration and DEP‐induced human platelet aggregation was inhibited by an NO donor.Conclusions and ImplicationsPulmonary exposure to DEP and subsequent platelet activation may contribute to the reports of increased cardiovascular risk, associated with exposure to airborne pollution, independent of its effects on inflammation or NO bioavailability.

Nitrative DNA damage induced by carbon-black nanoparticles in macrophages and lung epithelial cells

Carbon black (CB) is a nanomaterial used mainly in rubber products. Exposure to CB by inhalation causes malignant lung tumors in experimental animals. CB inhalation may cause chronic inflammation in the respiratory system, leading to carcinogenesis, but the mechanism remains unclear. Reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells under inflammatory conditions, and resulting DNA damage may contribute to carcinogenesis. In this study, we performed immunocytochemistry to determine whether CB exposure induces formation of 8-nitroguanine (8-nitroG), a nitrative DNA lesion formed under inflammatory conditions, in RAW 264.7 macrophage and A549 lung epithelial cells. We compared the DNA-damaging effects of CB particles with primary diameter 56nm (CB56) and 95nm (CB95). Both types of CB induced 8-nitroG formation, mainly in the nucleus of RAW 264.7 and A549 cells, and CB95 tended to induce more 8-nitroG formation than did CB56. Flow cytometry revealed that CB95 generated larger amount of reactive oxygen species than did CB56 in RAW 264.7 cells. The Griess method showed that CB95 produced significantly larger amount of nitric oxide (NO) than did CB56. Flow cytometry showed that CB95 was more efficiently internalized into the cells than was CB56. The cellular uptake of CB and 8-nitroG formation in RAW 264.7 cells were reduced by monodansylcadaverine, an inhibitor of clathrin-mediated endocytosis, and by siRNA for Ctlc (clathrin heavy chain) gene. CB induces nitrative DNA damage in cultured cells, and clathrin-mediated endocytosis is involved, at least in part.

Inflammation and Vascular Effects after Repeated Intratracheal Instillations of Carbon Black and Lipopolysaccharide.

Inflammation and oxidative stress are considered the main drivers of vasomotor dysfunction and progression of atherosclerosis after inhalation of particulate matter. In addition, new studies have shown that particle exposure can induce the level of bioactive mediators in serum, driving vascular- and systemic toxicity. We aimed to investigate if pulmonary inflammation would accelerate nanoparticle-induced atherosclerotic plaque progression in Apolipoprotein E knockout (ApoE-/-) mice. ApoE -/- mice were exposed to vehicle, 8.53 or 25.6 μg nanosized carbon black (CB) alone or spiked with LPS (0.2 μg/mouse/exposure; once a week for 10 weeks). Inflammation was determined by counting cells in bronchoalveolar lavage fluid. Serum Amyloid A3 (Saa3) expression and glutathione status were determined in lung tissue. Plaque progression was assessed in the aorta and the brachiocephalic artery. The effect of vasoactive mediators in plasma of exposed ApoE-/- mice was assessed in aorta rings isolated from naïve C57BL/6 mice. Pulmonary exposure to CB and/or LPS resulted in pulmonary inflammation with a robust influx of neutrophils. The CB exposure did not promote plaque progression in aorta or BCA. Incubation with 0.5% plasma extracted from CB-exposed ApoE-/- mice caused vasoconstriction in aorta rings isolated from naïve mice; this effect was abolished by the treatment with the serotonin receptor antagonist Ketanserin. In conclusion, repeated pulmonary exposure to nanosized CB and LPS caused lung inflammation without progression of atherosclerosis in ApoE-/- mice. Nevertheless, plasma extracted from mice exposed to nanosized CB induced vasoconstriction in aortas of naïve wild-type mice, an effect possibly related to increased plasma serotonin.

Bio-effect of nanoparticles in the cardiovascular system.

Nanoparticles (NPs; < 100 nm) are increasingly being applied in various fields due to their unique physicochemical properties. The increase in human exposure to NPs has raised concerns regarding their health and safety profiles. The potential correlation between NP exposure and several cardiovascular (CV) events has been demonstrated. The aim of this review is to provide a comprehensive evaluation of the current knowledge regarding the bio-toxic impacts of titanium oxide, silver, silica, carbon black, carbon nanotube, and zinc oxide NPs exposure on the CV system in terms of in vivo and in vitro experiments, which is not fully understood presently. Moreover, the potential toxic mechanisms of NPs in the CV system that are still being questioned are elaborately discussed, and the underlying capacity of NPs used in medicine for CV events are summarized. It will be an important instrument to extrapolate relevant data for human CV risk evaluation and management. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2881-2897, 2016.

Impact of Silver and Carbon Nanoparticle Exposures on Macrophage Responses to Mycobacterium tuberculosis (M.tb)

Abstract To assess biological risks of nanoparticle (NP) exposures, we examined the effects of silver (Ag) core Ag20-citrate (Ag20) and carbon black (CB) on human host innate immune responses to M.tb, a respiratory pathogen that causes tuberculosis. Human peripheral blood monocyte-derived macrophages (MDMs) were exposed to 1, 10, 25 and 50 μg/mL of Ag20 and CB for 4 and 24 h. At 4 h no significant cytotoxicity was observed with neither of the NPs at none of the concentrations examined relative to unexposed MDMs. At 24 h, viability of the MDMs was reduced by 60–70% and 20% following Ag20 and CB exposure, respectively, at doses ≥25 μg/mL. M.tb-induced host immune responses are mediated by the activation of TLR signalling pathways that culminate in NF-κB activation. Effects of Ag20 and CB (10μg/mL for 4 h, a nontoxic dose) on TLR signalling in M.tb-infected and uninfected MDMs were compared using a human TLR signaling pathway-specific profiler array. Ag20 exposure suppressed the M.tb-induced expression of a subset of NF-κB-mediated target genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). Ag20-mediated suppression of M.tb-induced activation of the TLR-signalling pathway was not due to decreased uptake of M.tb by MDMs or mycobacteriocidal effects of Ag20. Ag20 exposure increased the expression of HSPA1A mRNA that encodes for the stress-induced Hsp72. In contrast, exposure to CB resulted in little effects on M.tb-induced host gene expression indicating that immunosuppressive effects of Ag20 are NP-specific. Since Hsp72 has been shown to suppress NF-κB activation, we propose that Ag20-mediated Hsp72 upregulation contributes to the suppression of M.tb-induced NF-κB activation and host immune responses. Funding NIEHS 5U19 ES019536

Biocompatible Label-Free Detection of Carbon Black Particles by Femtosecond Pulsed Laser Microscopy.

Although adverse health effects of carbon black (CB) exposure are generally accepted, a direct, label-free approach for detecting CB particles in fluids and at the cellular level is still lacking. Here, we report nonincandescence related white-light (WL) generation by dry and suspended carbon black particles under illumination with femtosecond (fs) pulsed near-infrared light as a powerful tool for the detection of these carbonaceous materials. This observation is done for four different CB species with diameters ranging from 13 to 500 nm, suggesting this WL emission under fs near-infrared illumination is a general property of CB particles. As the emitted radiation spreads over the whole visible spectrum, detection is straightforward and flexible. The unique property of the described WL emission allows optical detection and unequivocal localization of CB particles in fluids and in cellular environments while simultaneously colocalizing different cellular components using various specific fluorophores as shown here using human lung fibroblasts. The experiments are performed on a typical multiphoton laser-scanning microscopy platform, widely available in research laboratories.

Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures

This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. Biological significanceIn this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.

Meta-Analysis of Cardiac Mortality in Three Cohorts of Carbon Black Production Workers.

Epidemiological studies have demonstrated associations between airborne environmental particle exposure and cardiac disease and mortality; however, few have examined such effects from poorly soluble particles of low toxicity such as manufactured carbon black (CB) particles in the work place. We combined standardised mortality ratio (SMR) and Cox proportional hazards results from cohort studies of US, UK and German CB production workers. Under a common protocol, we analysed mortality from all causes, heart disease (HD), ischemic heart disease (IHD) and acute myocardial infarction (AMI). Fixed and random effects (RE) meta-regression models were fit for employment duration, and for overall cumulative and lugged quantitative CB exposure estimates. Full cohort meta-SMRs (RE) were 1.01 (95% confidence interval (CI) 0.79–1.29) for HD; 1.02 (95% CI 0.80–1.30) for IHD, and 1.08 (95% CI 0.74–1.59) for AMI mortality. For all three outcomes, meta-SMRs were heterogeneous, increased with time since first and time since last exposure, and peaked after 25–29 or 10–14 years, respectively. Meta-Cox coefficients showed no association with lugged duration of exposure. A small but imprecise increased AMI mortality risk was suggested for cumulative exposure (RE-hazards ratio (HR) = 1.10 per 100 mg/m3-years; 95% CI 0.92–1.31), but not for lugged exposures. Our results do not demonstrate that airborne CB exposure increases all-cause or cardiac disease mortality.

Cohort Study of Carbon Black Exposure and Risk of Malignant and Nonmalignant Respiratory Disease Mortality in the US Carbon Black Industry.

To evaluate lung cancer and respiratory disease mortality associations with cumulative inhalable carbon black exposure among 6634 US carbon black workers. This analysis was performed using standardized mortality ratio (SMRs) and Cox regression analyses. Lung cancer mortality was decreased overall (SMR = 0.77; 95% confidence interval [CI], 0.67 to 0.89) but less so among hourly male workers (SMR = 0.87; 95% CI, 0.71 to 1.05). No exposure-response association was observed with time-dependent cumulative inhalable carbon black: hazard ratio [HR] = 1.0 (95% CI, 0.6 to 1.6) for 20 to less than 50 mg/m·yr); HR = 1.3 (95% CI, 0.8 to 2.1) for 50 to less than 100 mg/m·yr; and HR = 1.4 (95% CI, 0.9 to 2.1) for 100 mg/m·yr or more compared with referent (<20 mg/m·yr). No consistent associations were observed between cumulative inhalable carbon black exposure and respiratory disease mortality. Quantitative carbon black exposure estimates were not related to lung cancer or nonmalignant respiratory disease mortality.

Abstract 839: Effects of occupational exposure to carbon black on peripheral white blood cell counts

Abstract Carbon black (CB) is a material produced by the combustion of hydrocarbon gases or liquids and widely used in various industrial settings. The International Agency for Research on Cancer has classified CB as possibly carcinogenic to humans. Previous studies suggest that CB might induce lung inflammation and injury and it is well known that inflammation can play an important role in tumor development. However, the relationship between CB exposure and peripheral white blood cell counts is still not clear. To address this issue, we conducted a cross-sectional molecular epidemiology study of 106 CB-exposed male workers responsible for packing CB in a CB manufacturing facility that produced very high purity CB and 112 unexposed male control workers from the same geographic region who were frequency-matched to the exposed workers by age. The average number of years worked in the factory for the exposed workers was 12.5 years. Peripheral blood samples were collected and a complete blood cell count (CBC) and lymphocyte subset analysis were conducted. There was a borderline significant increase in eosinophil count, and a significant decrease in total lymphocyte and NK cell counts, among the exposed vs. control workers. Among non-smokers (35 exposed and 29 controls), CB exposure was associated with a borderline significant increase in total white blood cell and neutrophil counts, a significant increase in eosinophil counts, and a significant decrease in total lymphocyte, CD3+ T cell, CD8+ T cell and NK cell counts. There were no significant differences between exposed and control workers for any white blood cell measure among smokers. Also, there was some evidence of an interaction between carbon black exposure and smoking status for neutrophil, total lymphocyte, CD3+ T cell, and CD8+ T cell counts. Our results suggest that CB exposure may be associated with altered levels of cells that play an important role in the inflammatory process among non-smokers. Also, there was evidence for an interaction between CB exposure and tobacco use. These findings may provide new insights into particulate-associated disease. Citation Format: Yufei Dai, Rong Zhang, Yong Niu, Huawei Duan, Tao Meng, Meng Ye, Meili Shen, Ping Bin, Shanfa Yu, Roel Vermeulen, Nathaniel Rothman, Qing Lan, Yuxin Zheng. Effects of occupational exposure to carbon black on peripheral white blood cell counts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 839. doi:10.1158/1538-7445.AM2015-839