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Questions exist concerning the potential carcinogenic effects after welding fume exposure. Welding processes that use stainless steel (SS) materials can produce fumes that may contain metals (e.g., Cr, Ni) known to be carcinogenic to humans. The objective was to determine the effect of in vitro and in vivo welding fume treatment on free radical generation, DNA damage, cytotoxicity and apoptosis induction, all factors possibly involved with the pathogenesis of lung cancer. SS welding fume was collected during manual metal arc welding (MMA). Elemental analysis indicated that the MMA-SS sample was highly soluble in water, and a majority (87%) of the soluble metal was Cr. Using electron spin resonance (ESR), the SS welding fume had the ability to produce the biologically reactive hydroxyl radical (*OH), likely as a result of the reduction of Cr(VI) to Cr(V). In vitro treatment with the MMA-SS sample caused a concentration-dependent increase in DNA damage and lung macrophage death. In addition, a time-dependent increase in the number of apoptotic cells in lung tissue was observed after in vivo treatment with the welding fume. In summary, a soluble MMA-SS welding fume was found to generate reactive oxygen species and cause DNA damage, lung macrophage cytotoxicity and in vivo lung cell apoptosis. These responses have been shown to be involved in various toxicological and carcinogenic processes. The effects observed appear to be related to the soluble component of the MMA-SS sample that is predominately Cr. A more comprehensive in vivo animal study is ongoing in the laboratory that is continuing these experiments to try to elucidate the potential mechanisms that may be involved with welding fume-induced lung disease.

Arsenite is widely distributed environmental toxicant in water, food and air. It is a known human carcinogen, which is strongly associated with human cancers originated from liver, nasal cavity, lung, skin, bladder, kidney, and prostate. In this study, we investigated whether arsenite induces expression of hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric basic helix-loop-helix transcription factor, composed of HIF-1alpha and HIF-1beta/ARNT subunits; and is involved in tumor growth and angiogenesis. Here we demonstrate that arsenite induces the expression of HIF-1alpha but not HIF-1beta subunit in DU145 human prostate carcinoma cells. Arsenite also increases the expression of VEGF through the induction of HIF-1. We also found that arsenite activates PI3K and Akt that are required for arsenite-induced expression of HIF-1alpha and VEGF. The induction of HIF-1 and VEGF by arsenite can not be inhibited by MAP kinase inhibitors. Arsenite causes production of reactive oxygen species (ROS). The major species of ROS required for the induction of HIF-1 and VEGF is H2O2. These data indicate that the arsenite-induced activation of PI3K/Akt signaling and the expression of HIF-1 and VEGF through the generation of ROS could be an important mechanism in the arsenite-induced carcinogenesis.

A wide variety of metals have been reported to act as mutagenic and carcinogenic agents in both human and animal studies. The underlying mechanisms are being extensively investigated. Recently, a new sub-discipline of molecular carcinogenesis has surfaced and new techniques and instruments are being developed which allow exploration of the complex biological relationships and signaling pathways involved in response to metal exposure at the molecular level. The 2nd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis was held at NIOSH in Morgantown, West Virginia, Sept. 8-11, 2002. One hundred thirty scientist from sixteen countries presented their novel findings and investigations of metal-induced carcinogenesis. The conference focused on state-of-the-art research and developments in metal toxicity and carcinogenesis. Emphasis was placed on delineating molecular mechanisms involved in free radical effects, cellular uptake, signaling pathways/interaction, dose response, biomarkers, and resistance mechanisms. This article reviews some of the novel information presented at the conference and discusses future avenues of research in this field.

Exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate (PbCrO4), has been associated with lung cancer and respiratory tract toxicity. Previous studies indicate that the solubility of Cr(VI)-compounds is an important factor in Cr(VI)-induced carcinogenesis. The present study investigates reactive oxygen species (ROS) generation by PbCrO4 particles and cellular responses using RAW 264.7 cells. A mixture containing PbCrO4 and RAW 264.7 cells generated hydroxyl radical ((.)OH), using cellularly generated H2O2 as a precursor, as measured by electron spin resonance (ESR) spin trapping in combination with H2O2 and (.)OH scavengers, catalase and sodium formate. The effect of ascorbic acid on (.)OH radicals was also measured using ESR. Confocal microscopy showed that particles could become either bound to the cell surface or engulfed over a 120 min time period. H2O2 generation and O2 consumption were also increased after treatment of the cells with PbCrO4. Both NF-kappaB and AP-1 were activated after exposure to PbCrO4 particles as measured by the NF-kappaB or AP-1 luciferase reporter plasmid assay. Our investigation thus demonstrated that the RAW 264.7 cells phagocytized the PbCrO4 particles leading to accumulation of the particles within vacuoles in the cytoplasm. These particles could induce chronic production of ROS and activation of NF-kappaB and AP-1. Such induction of transcription pathways may be involved in the inflammatory and carcinogenic responses induced by Cr(VI)-containing particles.

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.

Residual oil fly ash (ROFA) is a particulate pollutant comprised of soluble and insoluble metals and is produced by the combustion of fossil fuels. The objective was to examine the pulmonary responses to chemically distinct ROFA samples collected from either a precipitator or air heater within the same power plant. The collected ROFA samples were suspended in saline (total sample), incubated for 24 h at 37 degrees C, centrifuged, separated into soluble and insoluble fractions, and the metal composition was determined. In addition, electron spin resonance (ESR) was used to detect short-lived free radical intermediates produced by the ROFA samples and the different fractions. On day 0, Male Sprague-Dawley rats were intratracheally instilled with saline (vehicle control) or the ROFA samples (1 mg/100 g body wt). At day 1, bronchoalveolar lavage was performed, and lung inflammation was assessed. On day 3, additional rats that had been treated with ROFA were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes, and pulmonary bacterial clearance was measured at days 6, 8, and 10. The precipitator ROFA was found to be more soluble and acidic with a significantly greater mass of each metal compared with the air heater ROFA. A prominent hydroxyl radical signal was measured for the total and soluble precipitator ROFA after the addition of H2O2, whereas the air heater ROFA and its fractions did not produce a signal. Precipitator ROFA induced a greater inflammatory response than air heater ROFA illustrated by a significant elevation in lung neutrophils. In addition, pulmonary clearance of L. monocytogenes was greatly diminished in the rats treated with the soluble and total precipitator ROFA samples. None of the air heater ROFA samples had an effect on lung bacterial clearance. In conclusion, precipitator ROFA, particularly the soluble fraction, generated a metal-dependent hydroxyl radical as measured by ESR and was shown to cause more inflammation and result in reduced lung defense against infection compared with air heater ROFA. These results are most likely due to differences in metal composition and solubility of the ROFA samples.

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.

The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal C141 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H2O2 and O2. fluorescence staining assay. Treatment of cells with UV radiation or H2O2 also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70(S6k) in mouse epidermal JB6 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70(S6k), while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (O2-. radical scavenger) did not inhibit any of these pathways. These results demonstrate that H2O2 generation is required for UV-induced phosphorylation of Akt and p70(S6k), and involved in activation of JNKs and p38 kinase, but not Erks.

While Cr (VI)-containing compounds are well established carcinogens, the mechanisms of their action remain to be investigated. In this study we show that Cr (VI) causes increased tyrosine phosphorylation in human lung epithelial A549 cells in a time-dependent manner. N-acetyl-cysteine (NAC), a general antioxidant, inhibited Cr (VI)-induced tyrosine phosphorylation. Catalase, a scavenger of H2O2, sodium formate and aspirin, scavengers of hydroxyl radical (*OH), also inhibited the increased tyrosine phosphorylation induced by Cr (VI). SOD, an inhibitor of superoxide radical (O2*-), caused less inhibition. ESR study shows that incubation of Cr (VI) with the A549 cells generates *OH radical. The generation of radical was decreased by addition of catalase and sodium formate, while SOD did not have any inhibitory effect. Oxygen consumption measurements show that addition of Cr (VI) to A549 cells resulted in enhanced molecular oxygen consumption. These results indicate that Cr (VI) can induce an increase in tyrosine phosphorylation. H2O2 and *OH radicals generated during the process are responsible for the increased tyrosine phosphorylation induced by Cr (VI).

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

Cr (VI) compounds are widely used in industries and are recognized human carcinogens. The mechanism of carcinogenesis associated with these compounds is not well understood. The present study focused on Cr (VI)-induced cell growth arrest in human lung epithelial A549 cells, using flow cytometric analysis of DNA content. Treatment of the cells with Cr (VI) at 1 microM caused a growth arrest at G2/M phase. An increase in Cr (VI) concentration enhanced the growth arrest. At a concentration of 25 microM, Cr (VI)-induced apoptosis became apparent. Superoxide dismutase (SOD) or sodium formate did not alter the Cr (VI)-induced cell growth arrest. While catalase inhibited growth, indicating H2O2 is an important mediator in Cr (VI)-induced G2/M phase arrest. Electron spin resonance (ESR) spin trapping measurements showed that incubation of cells with Cr (VI) generated hydroxyl radical (*OH). Catalase inhibited the *OH radical generation, indicating that H2O2 was generated from cells stimulated by Cr (VI), and that H2O2 functioned as a precursor for *OH radical generation. The formation of H2O2 from Cr (VI)-stimulated cells was also measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. The mechanism of reactive oxygen species generation involved the reduction of molecular oxygen as shown by oxygen consumption assay. These results support the following conclusions: (a) Reactive oxygen species are generated in Cr (VI)-stimulated A549 cells through reduction of molecular oxygen, (b) Among the reactive oxygen species generated, H2O2 played a major role in causing G2/M phase arrest in human lung epithelial cells.

Electron spin resonance (ESR) spin trapping was utilized to investigate the scavenging effects on hydroxyl radicals (*OH) and superoxide radicals (O2*-) by (-)-epigallocatechin-3-gallate (EGCG), one of the major anticancer compounds in tea. The spin trap used was 5,5-dimethyl-pyrroline N-oxide (DMPO). The Fenton reaction (Fe2+ + H2O2-->Fe3+ + *OH + OH-) was used as a source of *OH radicals. EGCG efficiently scavenges *OH radicals with reaction rate of 4.62 x 10(11) M(-1)sec(-1), which is an order of magnitude higher than several well recognized antioxidants, such as ascorbate, glutathione and cysteine. It also scavenges O2*- radicals as demonstrated by using xanthine and xanthine oxidase system as a source of O2*- radicals. Through its antioxidant properties, EGCG exhibited a protective effect against DNA damage induced by Cr(VI). EGCG also inhibited activation of nuclear transcription factor NF-kappaB induced by Cr(IV) and 12-o-tetradecanoylphorbol-13-acetate (TPA). The present studies provide a mechanistic basis for the reported anticarcinogenic properties of EGCG and related tea products.

Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process.

Electron spin resonance (ESR) was used to investigate the reaction of aspirin toward reactive oxygen species, such as hydroxyl radicals (*OH), superoxide radicals (O2-) and H2O2. The Fenton reaction (Fe(II) + H2O2 ---> FE(III) + *OH + OR) was used as a source of *OH radicals. The results show that aspirin is an efficient *OH radical scavenger with a reaction rate constant of k = 3.6 x 10(10) M(-1) sec(-1), which is faster than several well established antioxidants, such as ascorbate, glutathione and cysteine. However, aspirin is not a good scavenger for O2- or H2O2. Through its antioxidant property, aspirin exhibited a protective effect against silica-induced lipid peroxidation and DNA strand breakage. Aspirin also inhibited the activation of nuclear transcription factor-kappaB induced by silica, lipopolysaccharide or the transition metal, Fe(II), as demonstrated by electrophoretic mobility shift assay. The results show that aspirin functions as an antioxidant via its ability to scavenge *OH radicals. This antioxidant property may explain some of its various physiological and pharmacological actions.

Although it is generally believed that reactive oxygen species activate NF-kappaB, a primary oxidative stress-responsive transcription factor, it is unclear which one among these species causes NF-kappaB activation. Our hypothesis is that hydroxyl radical (*OH) functions as a messenger for the activation of NF-kappaB. Jurkat cells, macrophages and JB6 cells were used to test this hypothesis. Cr(VI), silica and ZnO were used as sources of *OH radicals. None of these *OH generating systems involves exogenous H2O2. Cr(VI) expressed enhanced activity in induction of NF-kappaB in Jurkat cells. This activation of NF-kappaB was decreased by a metal chelator, diethylene triaminepentaacetic acid or a H2O2 scavenger, catalase, but was increased by superoxide dismutase. Mn(II), which reacts with Cr(IV) to inhibit this metal ion-mediated *OH generation, decreased the NF-kappaB activation. Sodium formate, an *OH radical scavenger, also inhibited the NF-kappaB activation. Electron spin resonance measurements show that Cr(VI) was reduced by Jurket cells to Cr(IV) and Cr(V). During the reduction process, molecular oxygen was reduced to O2 and then to H2O2, which reacted with Cr(IV) and Cr(V) to generate *OH radical. The *OH generation correlated with the Cr(VI)-induced NF-kappaB activation. Similarly, silica caused NF-kappaB activation in macrophages via the *OH radical-mediated reaction. This radical was generated via metal mediated reaction from H2O2, which was generated by the reduction of molecular oxygen via O2- as an intermediate during the silica-stimulated 'respirable burst'. Silica particles did not cause *OH generation either in Jurket or in JB6 cells and thus did not cause any observable NF-kappaB activation in these cells. ZnO induced NF-kappaB activation in JB6 cells through the generation of *OH resulting from light irradiation of ZnO which was measured by electron spin resonance. The results thus show that *OH radical functions as a messenger for NF-kappaB activation. Antioxidants, which scavenge *OH radical or its precursors, inhibit NF-kappaB activation. Metal chelators, which make metal ions incapable of generating *OH from H2O2, inhibit activation of this transcription factor.