How does Integrative Oncology Influence Patients' Physical and Psychosocial Outcomes, and What are Patients, Carers and Healthcare Professionals' Experiences? An Integrative Review.

OBJECTIVE: To identify the relationship between integrative oncology (IO) services on patients' physical and psychosocial outcomes and to explore the experiences of IO among patients, carers and healthcare professionals. DATA SOURCES: This integrative review was reported according to PRISMA guidelines. A search architecture was developed using key words and the following databases were searched: Medline (OVID), EmCare for Nurses (OVID), PsycINFO (OVID); AMED (OVID), CINAHL (EBSCO), Pubmed, the Cochrane Library (CCRT and CDSR) controlled trials databases and ANZ CTR. All articles were assessed according to a pre-determined selection criterion. 426 articles were assessed and 18 were included (4 qualitative, 9 quantitative and 5 mixed methods). CONCLUSION: Patients reported a reduction in some cancer related symptoms and treatment related side effects. Positive psychosocial impacts were reported such as an increased ability to cope with their cancer diagnosis and treatment. The experiences of healthcare professionals highlighted the importance of a collaborative approach among the Multi-Disciplinary Team (MDT), ongoing education and research to ensure Complementary Integrative Therapies (CIT) were evidence-based. IMPLICATIONS FOR NURSING PRACTICE: The provision of IO impacts positively on patients' self-reported physical and emotional wellbeing and quality of life at all stages of their cancer experience. Patients reported that IO supported their engagement in their own health and wellbeing by increasing feelings of control and empowerment. However, to successfully integrate CIT with conventional cancer treatments it is imperative that cancer centres adopt a collaborative and evidence-based informed approach to CIT.

[Anal cancer: diagnostic and differential diagnostic issues]. / Das Analkanalkarzinom: Diagnostische und differenzialdiagnostische Aspekte.

Tumors of the anal canal are mostly epithelial in origin. The transition of gland-forming rectal mucosa via specialized urothelium-like cells at the dentate line to anal non-keratinized and finally perianal keratinized squamous epithelium implies a broad spectrum of tumor types, with most cancers exhibiting a mixture of different histological features. Moreover, secondary neoplasias extending into or metastasizing to the anal region need to be considered. Based on epithelial metaplasia at the transformation zone, poorly differentiated squamous anal carcinomas may show co-expression of both the squamous (CK5/6) and glandular type keratins (CK7). Since HPV infection of high-risk types (often 16 and 18) is etiologically associated with anal cancer, p16(INK4a) is highly sensitive and specific in the detection of high-grade anal squamous intraepithelial neoplasias (ASIN) and corresponding invasive squamous carcinomas. Diagnosis of secondary malignancies, including pagetoid extension into the anogenital region, requires the application of specific immunohistochemical marker panels.

The formation of reactive oxygen species catalyzed by neutral, aqueous extracts of NIST ambient particulate matter and diesel engine particles.

It is important to characterize the chemical properties of particulate matter in order to understand how low doses, inhaled by a susceptible population, might cause human health effects. The formation of reactive oxygen species catalyzed by neutral, aqueous extracts of two ambient particulate samples, National Institute of Standards & Technology (NIST) Standard Reference Materials (SRM) 1648 and 1649, and two diesel particulate samples, NIST SRM 1650 and SRM 2975, were measured. The formation of reactive oxygen species was estimated by measuring the formation of malondialdehyde from 2-deoxyribose in the presence of ascorbic acid; H2O2 was not added to this assay. SRM 1649, ambient particulate matter collected from Washington, DC, generated the most malondialdehyde, while SRM 2975, diesel particulate matter collected from a forklift, yielded the least amount. Desferrioxamine inhibited the formation of malondialdehyde from the particulate samples providing additional data to support the observation that transition metals were involved in the generation of reactive oxygen species. Six transition metal sulfates (iron sulfate, copper sulfate, vanadyl sulfate, cobalt sulfate, nickel sulfate, and zinc sulfate) were assayed for their ability to generate reactive oxygen species under the same conditions used for the particulate samples in order to facilitate comparisons between particles and these transition metals. The concentration of transition metals was measured in aqueous extracts of these particulate samples using ion-coupled plasma mass spectrometry (ICP-MS) analysis. There was qualitative agreement between the concentrations of Fe, Cu, and V and the amount of malondialdehyde produced from extracts of these particulate samples. These data suggest that transition metals can be dissolved from particles in neutral, aqueous solutions and that these metals are capable of catalyzing the formation of reactive oxygen species.

Using in vitro iron deposition on asbestos to model asbestos bodies formed in human lung.

Recent studies have shown that iron is an important factor in the chemical activity of asbestos and may play a key role in its biological effects. The most carcinogenic forms of asbestos, crocidolite and amosite, contain up to 27% iron by weight as part of their crystal structure. These minerals can acquire more iron after being inhaled, thereby forming asbestos bodies. Reported here is a method for depositing iron on asbestos fibers in vitro which produced iron deposits of the same form as observed on asbestos bodies removed from human lungs. Crocidolite and amosite were incubated in either FeCl(2) or FeCl(3) solutions for 2 h. To assess the effect of longer-term binding, crocidolite was incubated in FeCl(2) or FeCl(3) and amosite in FeCl(3) for 14 days. The amount of iron bound by the fibers was determined by measuring the amount remaining in the incubation solution using an iron assay with the chelator ferrozine. After iron loading had been carried out, the fibers were also examined for the presence of an increased amount of surface iron using X-ray photoelectron spectroscopy (XPS). XPS analysis showed an increased amount of surface iron on both Fe(II)- and Fe(III)-loaded crocidolite and only on Fe(III)-loaded amosite. In addition, atomic force microscopy revealed that the topography of amosite, incubated in 1 mM FeCl(3) solutions for 2 h, was very rough compared with that of the untreated fibers, further evidence of Fe(III) accumulation on the fiber surfaces. Analysis of long-term Fe(III)-loaded crocidolite and amosite using X-ray diffraction (XRD) suggested that ferrihydrite, a poorly crystallized hydrous ferric iron oxide, had formed. XRD also showed that ferrihydrite was present in amosite-core asbestos bodies taken from human lung. Auger electron spectroscopy (AES) confirmed that Fe and O were the only constituent elements present on the surface of the asbestos bodies, although H cannot be detected by AES and is presumably also present. Taken together for all samples, the data reported here suggest that Fe(II) binding may result from ion exchange, possibly with Na, on the fiber surfaces, whereas Fe(III) binding forms ferrihydrite on the fibers under the conditions used in this study. Therefore, fibers carefully loaded with Fe(III) in vitro may be a particularly appropriate and useful model for the study of chemical characteristics associated with asbestos bodies and their potential for interactions in a biosystem.

Mobilization of iron from coal fly ash was dependent upon the particle size and source of coal: analysis of rates and mechanisms.

The observed iron mobilization rate from size-fractionated coal fly ash is consistent with the model predictions for a limiting case of mass transfer where the dominant resistance is diffusion through a layer of depleted solid between the surface of spherical particles and a shrinking core of unreacted material. The rate of mobilization of iron from coal fly ash under physiologically relevant conditions in vitro was previously shown to depend on the size of the ash particles and on the source of the coal, and these in vitro measurements have been shown to correlate with indirect measurements of excess iron in cultured cells. Existing iron mobilization data were compared to mathematical models for mass transfer and chemical reaction in solid-liquid heterogeneous systems. Liquid-phase diffusion resistance can be ruled out as the rate-limiting mechanism for iron mobilization as the model predictions for this case are clearly inconsistent with the measurements. Other plausible hypotheses, such as a rate limited by a heterogeneous surface reaction, cannot be conclusively ruled out by the available data. These mathematical analysis methods are applicable to the design of future experiments to determine the rate-limiting mechanism for the mobilization of iron and of other transition metals from both ambient air samples and surrogates for major sources of particulate air pollution.

Mössbauer spectroscopy indicates that iron in an aluminosilicate glass phase is the source of the bioavailable iron from coal fly ash.

Iron speciation by Mössbauer spectroscopy indicates that ferric iron in an aluminosilicate glass phase is the source of the bioavailable iron in coal fly ash and that this iron species is associated with combustion particles, but not with crustal dust derived from soil minerals. Urban particulate has been shown to be a source of bioavailable iron and has been shown to be able to induce the formation of reactive species in cell culture experiments. Crustal dust and laboratory-generated coal fly ash have been studied as surrogates for two sources of metal-bearing particles in ambient air. As much as a 60-fold difference in the amount of iron mobilized by the chelator citrate was observed between fly ash and crustal dust samples with similar total iron contents. The extent of iron mobilization by citrate in vitro has been shown to correlate with indirect measures of excess iron in cultured cells and with assays for reactive oxygen species generation in vitro. Mössbauer spectroscopy of coal fly ash, before and after treatment with the chelator desferrioxamine B, showed that the iron in an aluminosilicate glass phase was preferentially removed. The removal of the glass-phase iron greatly reduced the amount of iron that could be mobilized by citrate and prevented the particles from inducing interleukin-8 in cultured human lung epithelial (A549) cells. Ferric iron in aluminosilicate glass is associated with particles formed at high temperatures followed by rapid cooling. The observation that ferric iron in aluminosilicate glass is the source of bioavailable iron in coal fly ash suggests that particles from ambient sources and other specific combustion sources should be examined for the presence of this potential source of bioavailable iron.

Interleukin-8 levels in human lung epithelial cells are increased in response to coal fly ash and vary with the bioavailability of iron, as a function of particle size and source of coal.

Particulate air pollution contains iron, and some of the pathological effects after inhalation may be due to radical species produced by iron-catalyzed reactions. We tested the hypothesis that iron present in coal fly ash (CFA) could induce the expression and synthesis of the inflammatory cytokine interleukin-8 (IL-8). CFA, containing as much as 14% iron, was used as a model combustion source particle. Three coal types were used to generate three size fractions enriched in particles [submicron (<1 micrometer), fine (<2.5 micrometer), or coarse (2.5-10 micrometer]), as well as the fraction of >10 micrometer. Treatment of human lung epithelial (A549) cells for 4 h with CFA from Utah enriched in <1 micrometer particles (20 microgram/cm(2)) resulted in a 2.6-fold increase in mRNA levels for IL-8. IL-8 levels were increased in the medium by as much as 8-fold when cells were treated with the fraction enriched in the smallest size Utah CFA for 24 h. IL-8 production was completely inhibited when the CFA was pretreated with the metal chelator desferrioxamine B, suggesting that a transition metal was responsible for the induction, probably iron. Treatment with a soluble form of iron, ferric ammonium citrate (FAC), mimicked the IL-8 level increase observed with CFA. There was a direct relationship, above a threshold level of bioavailable iron, between the levels of IL-8 and bioavailable iron in A549 cells treated with CFA or FAC. Further, the relationship between IL-8 and bioavailable iron for CFA was indistinguishable from that for FAC. These results strongly suggest that iron can induce IL-8 in A549 cells and that iron was the likely component of CFA that induced IL-8. CFA-induced IL-8 production was inhibited by tetramethylthiourea or dimethyl sulfoxide, suggesting that radical species were involved in the induction. These results demonstrate that iron present in CFA may be responsible for production and release of inflammatory mediators by the lung epithelium through generation of radical species and suggest that iron may contribute to the exacerbation of respiratory problems by particulate air pollution.

Bioavailability of iron from coal fly ash: mechanisms of mobilization and of biological effects.

Particulate air pollution contains iron that may be involved in the pathological effects after inhalation. This article reviews work demonstrating that ambient particulate samples (Standard Reference Material [SRM] 1648 and SRM 1649, from the National Institute of Science and Technology) contain iron that can be mobilized from the particle in vitro and inside human lung epithelial (A549) cells. The mobilized iron can then catalyze the formation of reactive oxygen species (ROS). Work is also reviewed on the generation and size fractionation of coal fly ash (CFA) from three commercially important coal types, as well as size fractionation of three types of noncombustion particles. The availability of iron from these particles to A549 cells was measured by citrate mobilization in vitro and induction of the iron storage protein ferritin in particle-treated cells. The amount of bioavailable iron decreased with increasing particle size. The ability of particles to induce synthesis of the proinflammatory cytokine interleukin-8 (IL-8) was also determined. As with the bioavailability of iron, there was an inverse correlation with size. Further work showed that iron in CFA is responsible for IL-8 induction. Mössbauer spectroscopy of a CFA sample before and after desferrioxamine B treatment to remove bioavailable iron showed that the bioavailable iron was associated with the glassy aluminosilicate fraction of the particle. In conclusion, this work shows that bioavailable iron is responsible for ROS production by SRMs and IL-8 induction by CFA in A549 cells. The source of this bioavailable iron in CFA is glassy aluminosilicates, which are found at higher levels in smaller sizes of CFA.

Mechanisms of DNA oxidation.

Oxidative damage of DNA caused by a variety of chemical and physical agents appears to be linked to cancer. However, it is becoming increasingly clear that endogenous generation of oxidants, such as hydroxyl radical and peroxynitrite, lead to oxidation of DNA, and this may cause cancer in individuals where no obvious exposure to chemical or physical agents known to be carcinogenic has occurred. The mechanisms for generation of these two oxidants in living organisms will be discussed and their reactivities with DNA to produce oxidized products (e.g., 8-oxo-dG) will be presented with special emphasis on the individual characteristics of the generation and reactivity of each oxidant.

In vitro effects of coal fly ashes: hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells.

Oxygen radical generation due to surface radicals, inflammation, and iron release has been suggested as the mechanism of adverse effects of quartz, such as emphysema, fibrosis, and carcinogenic effects. Therefore, we measured iron release, acellular generation of hydroxyl radicals, and oxidative DNA damage and cytotoxicity in rat lung epithelial (RLE) cells by different coal fly ashes (CFA) that contain both quartz and iron. Seven samples of CFA with different particle size and quartz content (up to 14.1%) were tested along with silica (alpha-quartz), ground coal, and coal mine dust (respirable) as positive control particles, and fine TiO(2) (anatase) as a negative control. Five test samples were pulverized fuel ashes (PFA), two samples were coal gasification (SCG) ashes (quartz content <0.1%), and one sample was a ground coal. No marked differences between SCG and PFA fly ashes were observed, and toxicity did not correlate with physicochemical characteristics or effect parameters. Stable surface radicals were only detected in the reference particles silica and coal mine dust, but not in CFA. On the other hand, hydroxyl radical generation by all fly ashes was observed in the presence of hydrogen peroxide, which was positively correlated with iron mobilization and inhibited by deferoxamine, but not correlated with iron or quartz content. Also a relationship between acellular hydroxyl radical generation and oxidative DNA damage in RLE cells by CFA was observed. Differences in hydroxyl radical generation and oxidative damage by the CFA were not related to iron and quartz content, but the respirable ashes (MAT023, 38, and 41) showed a very extensive level of hydroxyl radical generation in comparison to nonrespirable fly ashes and respirable references. This radical generation was clearly related to the iron mobilization from these particles. In conclusion, the mechanisms by which CFA and the positive references (silica, coal mine dust) affect rat lung epithelial cells seem to be different, and the data suggest that quartz in CFA does not act the same as quartz in silica or coal mine dust. On the other hand, the results indicate an important role for size and iron release in generation and subsequent effects of reactive oxygen species caused by CFA.

Mobilization of iron from coal fly ash was dependent upon the particle size and the source of coal.

Particulate air pollution, including coal fly ash, contains iron, and some of the pathological effects after inhalation may be due to reactive oxygen species produced by iron-catalyzed reactions. The objective of this study was to determine whether iron, present in coal fly ash, was mobilized, leading to ferritin induction in human airway epithelial cells, and whether the size of the particles affected the amount of iron mobilized. Three types of coal were used to generate the three size fractions of fly ash collected. The Utah coal fly ash was generated from a bituminous b coal, the Illinois coal fly ash from a bituminous c coal, and the North Dakota coal fly ash from a lignite a coal. Three size fractions were studied to compare the amount of iron mobilized in human airway epithelial (A549) cells and by citrate in cell-free suspensions. The size fractions selected were fine (<2.5 microm) and coarse (2.5-10 microm) components of PM10, airborne particulate matter <10 microm in diameter, and the fraction greater than 10 microm. Coal fly ash samples were incubated with 1 mM citrate to determine if iron associated with coal fly ash could be mobilized. Iron was mobilized by citrate from all three size fractions of all three coal types to levels as high as 56.7 nmol of Fe/mg of coal fly ash after 24 h. With all three coal types, more iron was mobilized by citrate from the <2.5 microm fraction than from the >2.5 microm fractions. Further, the mobilized iron was in the Fe(III) form. To determine if iron associated with the coal fly ash could be mobilized by A549 cells, cells were treated with coal fly ash, and the amount of the iron storage protein ferritin was determined after 24 h. Ferritin levels were increased by as much as 11.9-fold in cells treated with coal fly ash. With two of the three types of coal studied, more ferritin was induced in cells treated with the <2.5 microm fraction than with the >2.5 microm fractions. Further, inhibition of the endocytosis of the coal fly ash by the cells resulted in ferritin levels that were near that of the untreated cells, suggesting that iron was mobilized intracellularly, not in the culture medium. The results of this study suggest that differences in particle size and speciation of iron may affect the release of iron in human airway epithelial cells.

Regulation of nitric oxide synthase induction by iron and glutathione in asbestos-treated human lung epithelial cells.

Treatment of human lung epithelial (A549) cells with crocidolite asbestos resulted in the induction of the inducible form of nitric oxide synthase (iNOS), production of NO, and a dramatic decrease in intracellular reduced glutathione (GSH). Iron, mobilized from the crocidolite fibers (27% iron by weight), and the formation of NO were required for the formation of 2'-deoxy-7-hydro-8-oxoguanosine in the DNA of the A549 cells, but not for the decrease in GSH. Therefore, we investigated the role of GSH and iron in the induction of iNOS in A549 cells by crocidolite. Iron was required for the induction of iNOS by crocidolite. A fivefold higher amount of chrysotile asbestos (3% iron by weight) was required to cause a similar decrease in intracellular GSH and induction of iNOS. In the absence of asbestos, treatment with either buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, or ferric ammonium citrate (FAC), a soluble form of iron, did not result in induction of iNOS. However, iNOS was induced when A549 cells were treated simultaneously with BSO and FAC. The presence of 5 mM N-acetylcysteine prevented induction of iNOS in crocidolite-treated A549 cells. These observations suggest that the induction of iNOS resulted from a decrease in intracellular GSH and the presence of iron from the asbestos fibers.

Participation of iron and nitric oxide in the mutagenicity of asbestos in hgprt-, gpt+ Chinese hamster V79 cells.

Crocidolite asbestos is known to cause cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The mechanism responsible for the carcinogenicity of asbestos is not known. Iron associated with asbestos is thought to play a role by catalyzing the formation of reactive oxygen species, which may cause DNA damage, leading to mutations and cancer. Here, we examined whether asbestos can induce mutations in Chinese hamster hgprt+ V79 cells or transgenic hgprt-, gpt+ V79 cells (G12). Treatment with 6 microg/cm2 crocidolite for 24 h caused a 2-fold increase in the mutation frequency at the gpt locus of G12 cells, but no increase at the hgprt locus of V79 cells. The mutation frequency at the gpt locus of G12 cells increased with increasing treatment dose of crocidolite. The mutations induced by crocidolite appeared to be due to the generation of reactive oxygen species catalyzed by iron associated with the fibers, because treatment of G12 cells in iron-free medium with fibers from which redox active iron had been removed with desferrioxamine B prevented all of the gpt- mutations above untreated control levels. In addition, treatment of cells with a soluble form of iron, 1.5 mM ferric ammonium citrate, resulted in an increase in mutation frequency at the gpt locus of approximately 1.5 fold above that of untreated G12 cells with no increase in mutations at the hgprt locus of V79 cells with ferric ammonium citrate. We also investigated the effect of nitric oxide on the mutagenicity of crocidolite in G12 cells. When G12 cells were treated with 3 microg/cm2 of crocidolite in the presence of nitric oxide-generating compound, 200 microM diethyltriamine/NO, the mutation frequency increased to a level that was more than additive for crocidolite or diethyltriamine/NO treatment alone. These results strongly suggest that the presence of iron and nitric oxide may either lead to the generation of another reactive, mutagenic species, such as peroxynitrite, or that nitric oxide inhibits a DNA repair enzyme(s), leading to an increase in mutations.

Efflux of reduced glutathione after exposure of human lung epithelial cells to crocidolite asbestos.

This study investigated glutathione (GSH) homeostasis in human lung epithelial cells (A549) exposed to crocidolite. Exposure of A549 cells to 3 micrograms/cm2 crocidolite resulted in a decrease in intracellular reduced glutathione by 36% without a corresponding increase in GSH disulfide. After a 24-hr exposure to crocidolite, 75% of the intracellular GSH lost was recovered in the extracellular medium, of which 50% was in reduced form. Since the half-life of reduced GSH in culture medium was less than 1 hr, this suggests that reduced GSH was released continuously from the cells after treatment. The release of GSH did not appear to result from nonspecific membrane damage, as there was no concomitant release of lactate dehydrogenase or 14C-adenine from loaded cells after crocidolite treatment for 24 hr. Crocidolite exposure resulted in the formation of S-nitrosothiols but no increase in the level of GSH-protein mixed disulfides or GSH conjugates. Exposure of A549 cells to crocidolite for 24 hr decreased gamma glutamylcysteine synthetase (gamma-GCS) activity by 47% without changes in the activities of GSH reductase, GSH peroxidase, GSH S-transferase, or glucose-6-phosphate dehydrogenase. Treatment of cells with crocidolite pretreated with the iron chelator desferrioxamine B resulted in the same level of intracellular GSH depletion and efflux and the same decrease in gamma-GCS activity as treatment with unmodified crocidolite, which suggests that iron-catalyzed reactions were not responsible for the GSH depletion.

Mobilization of iron from urban particulates leads to generation of reactive oxygen species in vitro and induction of ferritin synthesis in human lung epithelial cells.

Many of the biochemical effects of asbestos in cultured cells have been shown to be due to iron, which can be as high as 27% by weight. Urban air particulates also contain iron, and some of the pathological effects after inhalation may be due to reactive oxygen species produced by iron-catalyzed reactions. Two standard reference material (SRM) urban air particulate samples were used for the studies described here. SRM 1648 (3.9% iron by weight) was collected in the St. Louis, MO, area, and SRM 1649 (3% iron by weight) was collected in the Washington, DC, area. To determine if iron associated with urban particulates could be mobilized, as it is from asbestos, SRMs 1648 and 1649 were incubated with 1 mM citrate or EDTA, in the presence or absence of ascorbate. Iron was mobilized from both particulates by either chelator, especially in the presence of ascorbate. Citrate, in the presence of ascorbate, mobilized 30.9 nmol of Fe/mg of SRM 1648 and 65.1 nmol of Fe/mg of SRM 1649 in 24 h. EDTA, in the presence of ascorbate, mobilized 53.8 nmol of Fe/mg of SRM 1648 and 98.8 nmol of Fe/mg of SRM 1649 in 24 h. To determine whether reactive oxygen species were being produced by the particulate iron, each particulate was incubated with phi X174 RFI DNA in the presence or absence of ascorbate. Single-strand breaks (SSBs) were produced by either particulate, but only in the presence of ascorbate. Incubation of SRM 1648 or 1649 (0.5 mg/mL) with DNA in the presence of ascorbate and citrate resulted in 20% or 34% DNA with SSBs, respectively. Incubation of SRM 1648 or 1649 (0.1 mg/mL) with DNA in the presence of ascorbate and EDTA resulted in 26% or 45% DNA with SSBs, respectively. To determine if iron associated with urban particulates could be mobilized by human lung epithelial cells (A549), cells were treated with particulates and the amount of the iron storage protein ferritin was determined at the end of treatment. The 6.4- or 8.4-fold increase in ferritin observed in cells treated with SRM 1648 or 1649, respectively, over that of control (untreated) cells strongly suggested that iron was mobilized in the cultured cells. If similar mobilization and reactivity of the iron occurs in the lung, this may explain some of the pathological effects of urban particulates.

Induction of ferritin synthesis in human lung epithelial cells treated with crocidolite asbestos.

Crocidolite asbestos is a known human carcinogen containing 27% iron by weight. It has previously been shown that iron was mobilized intracellularly from crocidolite after treatment of human lung epithelial cells (A549) and that the toxicity of the fibers was directly related to how much mobilized iron was in the < 10,000 MW (low-molecular-weight, LMW) fraction [C. C. Chao, L.G. Lund, K. R. Zinn, and A. E. Aust (1994) Arch. Biochem. Biophys. 314, 384-391]. The data here show that iron mobilization from crocidolite began immediately after treatment of the A549 cells and increased linearly with time. However, the synthesis of ferritin, an iron storage protein, did not begin until after 4 h of treatment, reaching a sustained maximum after 12 h. Mobilized iron was preferentially incorporated into the nonferritin-protein fraction up to 7 h after treatment, when the amount of iron mobilized was low and before significant accumulation of newly synthesized ferritin had occurred. This suggested that these cultured cells needed additional iron for synthesis of iron-requiring proteins and that iron mobilized from crocidolite could be utilized directly for this purpose. Subsequent to this, additional mobilized iron was incorporated into newly synthesized ferritin. Even though iron from crocidolite was incorporated into newly synthesized ferritin or into other proteins, the amount of iron from crocidolite in the LMW fraction remained constant during the 24 h. Thus, it appeared that synthesis of ferritin may not have fully protected the cells from the toxic effects of iron mobilized from crocidolite.

Participation of nitric oxide and iron in the oxidation of DNA in asbestos-treated human lung epithelial cells.

Treatment of human lung epithelial (A549) cells with crocidolite resulted in the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the DNA, synthesis of mRNA for the inducible form of nitric oxide synthase (NOS), and increased intracellular nitrite (NO2-), a stable oxidation product of NO. Iron, associated with crocidolite, was involved in both NO2- and 8-OHdG formation. Addition of the NOS inhibitor, aminoguanidine (AG), reduced intracellular NO2- and prevented formation of 8-OHdG in crocidolite-treated cells, suggesting that NO was required in 8-OHdG formation. Addition of an NO-generating compound, diethyltriamine/NO, with AG and crocidolite resulted in recovery of 8-OHdG, further supporting a role for NO in oxidation of deoxyguanosine.

The effect of iron binding on the ability of crocidolite asbestos to catalyze DNA single-strand breaks.

Crocidolite or crocidolite pretreated with desferrioxamine-B (DF crocidolite) was exposed to ferrous chloride solutions to determine whether iron could be bound from solution. Native crocidolite was capable of binding up to 57 nmol Fe2+/mg fiber in 60 min, while the DF crocidolite was capable of binding only 5.5 nmol Fe2+/mg fiber. The rate of iron binding for the first 5 min of exposure was independent of the concentration of iron in the solution, suggesting that there was a group of rapidly saturable sites, approximately 1.5 x 10(18) binding sites/m2 crocidolite surface, which were responsible for the immediate binding. This process was followed by a slower binding phase, likely occurring at other sites. Crocidolite and DF crocidolite, with various amounts of iron bound, were assayed for their abilities to catalyze the formation of DNA single-strand breaks (SSBs) in phi X174 RFI DNA. Native crocidolite with additional iron bound did not significantly change in its ability to cause DNA SSBs in 15 or 30 min incubations, even though more iron could be mobilized from the iron-treated crocidolite at 4 or 24 h. DF crocidolite, after the addition of iron, had a significantly increased ability to form DNA SSBs. DF crocidolite with 0, 3.0 or 5.5 mmol Fe2+/mg catalyzed the formation of DNA SSBs in 21, 42 or 51% of the DNA respectively in the presence of EDTA and ascorbate. Fibers were also incubated in tissue culture medium with or without iron salts. The fibers incubated in the iron-containing medium had an increased ability to form DNA SSBs. These results suggest that fibers such as crocidolite may be capable of binding iron from intracellular sources. This additional iron may be as reactive as the intrinsic iron and may increase the reactive lifetime of the fiber.