Qamar Rahman

Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases ☆

Exposure to asbestos causes cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The pathogenesis of asbestos-related diseases is complicated and still poorly understood. Studies on animal models and cell cultures have indicated that asbestos fibers generate reactive oxygen and nitrogen species (ROS/RNS) and cause oxidation and/or nitrosylation of proteins and DNA. The ionic state of iron and its ability to be mobilized determine the oxidant-inducing potential of pathogenic iron-containing asbestos types. In addition to their capacity to damage macromolecules, oxidants play important roles in the initiation of numerous signal transduction pathways that are linked to apoptosis, inflammation, and proliferation. There is strong evidence supporting the premise that oxidants contribute to asbestos-induced lung injury; thus, strategies for reducing oxidant stress to pulmonary cells may attenuate the deleterious effects of asbestos.

Glutathione Redox System in Oxidative Lung Injury

Glutathione (GSH) is a ubiquitous intracellular thiol present in all tissues, including lung. Besides maintaining cellular integrity by creating a reduced environment, GSH has multiple functions, including detoxification of xenobiotics, synthesis of proteins, nucleic acids, and leukotrienes. Present in high concentrations in bronchoalveolar lavage fluid (BALF), GSH provides protection to the lung from oxidative injury induced by different endogenous or exogenous pulmonary toxicants. Its depletion in the lung has been associated with the increased risk of lung damage and disease. The redox system of GSH consists of primary and secondary antioxidants, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PD). Alterations in the activities of these enzymes may reflect reduced cellular defense and may serve as surrogate markers of many lung diseases. As GSH is also involved in the regulation of expression of protooncogenes and apoptosis (programmed cell death), the development of diseases such as cancer and human immune deficiency may be affected by depleting or elevating cellular GSH levels. Exogenous delivery of GSH or its precursor N-acetyl cysteine (NAC) is being used as chemotherapeutic approach.

Cytotoxicity, pro-oxidant effects and antioxidant depletion in rat lung alveolar macrophages exposed to ultrafine titanium dioxide.

In order to understand the pulmonary toxicity of ultrafine titanium dioxide (UF‐TiO2) particles, various biochemical and chemical parameters were assayed in rat alveolar macrophages (AMs) and cell‐free lavage fluid. Single intratracheal exposure of UF‐TiO2 (2 mg per rat) caused cytotoxicity to pulmonary AMs. An increase in the population of AMs could be observed, followed by increased activities of lactate dehydrogenase and acid phosphatase in cell‐free lavage fluid. In addition, AMs showed an adaptive response because the activities of glutathione peroxidase, glutathione reductase, glucose‐6‐phosphate dehydrogenase and glutathione S‐transferase were increased in these cells. However, this enhancement of antioxidant enzymes could not diminish the enhanced lipid peroxidation and increased rate of hydrogen peroxide generation. The level of glutathione remained decreased in UF‐TiO2‐exposed rat AMs. The data suggest that the induction of antioxidant enzymes by these cells for self‐protection is not sufficient to cope against the toxic action of UF‐TiO2, which may lead to oxidative stress.

Nanoparticles Induce Changes of the Electrical Activity of Neuronal Networks on Microelectrode Array Neurochips

Background Nanomaterials are extensively used in industry and daily life, but little is known about possible health effects. An intensified research regarding toxicity of nanomaterials is urgently needed. Several studies have demonstrated that nanoparticles (NPs; diameter < 100 nm) can be transported to the central nervous system; however, interference of NPs with the electrical activity of neurons has not yet been shown. Objectives/methods We investigated the acute electrophysiological effects of carbon black (CB), hematite (Fe2O3), and titanium dioxide (TiO2) NPs in primary murine cortical networks on microelectrode array (MEA) neurochips. Uptake of NPs was studied by transmission electron microscopy (TEM), and intracellular formation of reactive oxygen species (ROS) was studied by flow cytometry. Results The multiparametric assessment of electrical activity changes caused by the NPs revealed an NP-specific and concentration-dependent inhibition of the firing patterns. The number of action potentials and the frequency of their patterns (spike and burst rates) showed a significant particle-dependent decrease and significant differences in potency. Further, we detected the uptake of CB, Fe2O3, and TiO2 into glial cells and neurons by TEM. Additionally, 24 hr exposure to TiO2 NPs caused intracellular formation of ROS in neuronal and glial cells, whereas exposure to CB and Fe2O3 NPs up to a concentration of 10 μg/cm2 did not induce significant changes in free radical levels. Conclusion NPs at low particle concentrations are able to exhibit a neurotoxic effect by disturbing the electrical activity of neuronal networks, but the underlying mechanisms depend on the particle type.

Chrysotile-mediated imbalance in the glutathione redox system in the development of pulmonary injury.

A significant depletion in the content of glutathione (GSH) and alteration in GSH redox system enzymes were observed in the lung of chrysotile-exposed animals (5 mg) during different developmental stages of asbestosis. In the alveolar macrophages (AM) of exposed animals, the depletion in GSH started from day 1 and reached a maximum at day 16, whereas in lung tissue the maximum depletion was observed when fibrosis has matured. It appears that cellular GSH depletion triggers oxidative stress in the system as observed from increased thiobarbituric acid reactive substance (TBARS) production and alteration in the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD) and glutathione S-transferase (GST), the enzymes regulating oxidative tone. The depletion in GSH was also observed in red blood cells (RBC) of the exposed animals reaching a maximum when fibrosis matured. Thus the observed depletion in GSH, ascorbic acid and alteration in GSH redox system enzymes may be involved in fibrosis and carcinogenesis induced by chrysotile.

ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro

Asbestos is a known carcinogen and co-carcinogen. It is a persisting risk in our daily life due to its use in building material as asbestos-cement powder. The present study done on V79-cells (Chinese hamster lung cells) demonstrates the cytotoxic and genotoxic potential of asbestos-cement powder (ACP) in comparison with chrysotile asbestos. A co-exposure of chrysotile and ACP was tested using the cell viability test and the micronucleus assay. The kinetochore analysis had been used to analyse the pathway causing such genotoxic effects. Thiobarbituric acid-reactive substances were determined as evidence for the production of reactive oxygen species. Both, asbestos cement as well as chrysotile formed micronuclei and induced loss of cell viability in a concentration- and time- dependent way. Results of TBARS analysis and iron chelator experiments showed induction of free radicals in ACP- and chrysotile exposed cultures. CaSO4 appeared to be a negligible entity in enhancing the toxic potential of ACP. The co-exposure of both, ACP and chrysotile, showed an additive effect in enhancing the toxicity. The overall study suggests that asbestos-cement is cytotoxic as well as genotoxic in vitro. In comparison to chrysotile the magnitude of the toxicity was less, but co-exposure increased the toxicity of both.

Cyto-genotoxicity of amphibole asbestos fibers in cultured human lung epithelial cell line: role of surface iron.

The present investigations correlate the potentials of the reactive oxygen species (ROS) generation and the cyto-genotoxicity of amphibole asbestos fibers (amosite, crocidolite and tremolite) with their surface iron, under in vitro controlled conditions, using A549 cells (human lung epithelial cell line). The mobilizable surface iron was measured by Atomic Absorption Spectroscopy; the production of ROS was investigated using 2, 7 dichloro-dihydrofluorescein-diacetate (DCFH-DA) dye; for cytotoxicity assessment, the intracellular organelles specific damages were measured, using 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT) assay; and, the genotoxic potential of amphibole fibers was determined by cytokinesis block micronucleus (CBMN) assay. In the study, highest amount of ROS was generated by crocidolite followed by tremolite and minimum with amosite. In MTT assay, the time- and concentration-dependent decrease in percent cell viability was recorded with all the three amphibole fibers, tremolite being most cytotoxic, followed by crocidolite, and then amosite. In genotoxicity assay, an increase in the frequency of micronuclei (MNi) in binucleated (BN) cells was observed, where crocidolite was most genotoxic, followed by tremolite, and amosite the least.The comparison of results depicts a clear trend of cyto-genotoxic potential paralleling the ROS generation, suggesting a definite role of oxidative stress in fiber-induced toxicity. However, amosite contains maximum surface iron (28%), followed by crocidolite (27%), and tremolite carrying least (as contaminant) or no iron, the mobilizable surface iron is maximum in crocidolite followed by amosite and is minimum in tremolite. The mobilizable iron somewhat corresponds with the ROS generation capacity of these fibers. This shows that the surface iron could be mainly responsible for amphibole asbestos-induced ROS toxicity; though it may not be the only factor responsible, other factors like shape and size etc., also play role in amphibole asbestos-induced toxicity.

Frequency of sister chromatid exchange and chromosomal aberrations in asbestos cement workers

Exposure to asbestos minerals has been associated with a wide variety of adverse health effects including lung cancer, pleural mesothelioma, and cancer of other organs. It was shown previously that asbestos samples collected from a local asbestos factory enhanced sister chromatid exchanges (SCEs) and chromosomal aberrations in vitro using human lymphocytes. In the present study, 22 workers from the same factory and 12 controls were further investigated. Controls were matched for age, sex, and socioeconomic state. The peripheral blood lymphocytes were cultured and harvested at 48 hours for studies of chromosomal aberrations and at 72 hours for SCE frequency determinations. Asbestos workers had a raised mean SCE rate and increased numbers of chromosomal aberrations compared with a control population. Most of the chromosomal aberrations were chromatid gap and break types.

N-acetyl l-cysteine attenuates oxidant-mediated toxicity induced by chrysotile fibers

Chrysotile, an important commercial variety of asbestos, is known to cause oxidative stress by enhancing production of hydrogen peroxide (H(2)O(2)) and thiobarbituric acid reactive substances (TBARS), depleting glutathione (GSH) and altering levels of GSH redox system enzymes. N-acetyl L-cysteine (NAC), a compound that increases GSH levels, protects cells against chrysotile toxicity. In the present study, rats were exposed intratracheally to a single dose (5 mg/rat) of chrysotile. This was followed by a daily dose of NAC 50 mg/kg. b. wt., i.p. At 1, 4, 8 and 16 days post chrysotile exposure lung lavage fluid was collected to determine H(2)O(2) generation, TBARS production, GSH level and its redox system enzymes activities. A significant decrease in H(2)O(2) and TBARS, an increase in GSH content and its redox system enzymes was observed in chrysotile+NAC animals in comparison to chrysotile-exposed animals. In this preliminary study it appears that NAC may be protecting cells against oxidative damage. This protection may be due to its ability to maintain intracellular GSH/oxidative scavenging capability.

Activation of alveolar macrophages and peripheral red blood cells in rats exposed to fibers/particles.

The cytotoxic and oxidative responses of crocidolite and chrysotile asbestos fibers and ultrafine titanium dioxide (UF-TiO2) particles were measured in alveolar macrophages (AM) and peripheral red blood cells (RBC) of rat after 30 days with a single intratracheal exposure (5 mg). The following responses were observed one month after fiber/particle instillation: (1) AM population increased; (2) lactate dehydrogenase and acid phosphatase activities in cell free lung lavage fluid increased; (3) substances that react with hydrogen peroxide or thiobarbituric acid were elevated in both AM and peripheral RBC; (4) glutathione peroxidase, glutathione reductase, and catalase were altered in both AM and peripheral RBC; (5) glutathione and ascorbic acid decreased in both AM and peripheral RBC. A significant difference from negative controls was noted in all responses of the two fiber-exposed groups, and in most responses of the UF-TiO2-exposed group. The level of responses to the three test substances suggested a decreasing order of toxicity, with crocidolite > chrysotile > UF-TiO2.