RESUMEN

Patients with porphyria cutanea tarda (PCT) develop hepatocellular carcinoma as a late consequence. Pre-loading of C57BL/10ScSn mice with iron greatly sensitizes them to the induction of hepatic porphyria caused by hexachlorobenzene (HCB). HCB will also cause liver tumors in experimental animals. Elevated liver iron stores are implicated in the development of some human liver cancers in connection with its known catalytic role in generation of highly reactive activated oxygen species. The aim of this study was to determine the lipid and DNA oxidative damage in iron and HCB-induced porphyric mice. C57BL/10ScSn mice received i.p. injections of dextran sulfate (control), iron (Imferon) or combined iron and HCB. 6 weeks after treatment plasma ALT levels and hepatic free iron, porphyrin, lipid peroxides and 8-hydroxyguanosine (8-OHdG) levels were analyzed. Hepatic porphyrin level was significantly (p < 0.001) increased following combined iron/HCB treatment as compared to control mice. The level of lipid peroxides increased 9-fold (p = 0.001) and 35-fold (p < 0.001) after iron and iron/HCB treatment respectively, whereas the level of 8-OHdG was increased 2.5-fold (p = 0.002) and 7.5-fold (p < 0.001) after iron and iron/HCB treatment respectively as compared to control mice. The authors conclude that iron overload in conjugation with HCB induce lipid and DNA oxidative damage in C57BL/10ScSn mice. DNA oxidative damage may be important in the early events of hepatic carcinogenesis in experimental porphyria.

Asunto(s)

Daño del ADN/fisiología , Hemocromatosis/patología , Peroxidación de Lípido/fisiología , Neoplasias Hepáticas Experimentales/patología , Porfiria Cutánea Tardía/patología , Porfirias Hepáticas/patología , Animales , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Hemocromatosis/inducido químicamente , Hexaclorobenceno/toxicidad , Complejo Hierro-Dextran/toxicidad , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Porfiria Cutánea Tardía/inducido químicamente , Porfirias Hepáticas/inducido químicamente

RESUMEN

The effect of vitamin E treatment on total porphyrin content, lipid peroxidation (LOOH) and 8-hydroxydeoxyguanosine (8-OHdG) was studied in the livers of C57BL/10ScSn mice following hexachlorobenzene (HCB) and iron treatment. HCB was administered i.p. (totalling 300 mg/kg) twice, with 1 week interval. Three days after the first HCB injection iron-dextran was given i.p. (500 mg Fe per kg). Vitamin E was administered weekly (20 mg/kg) by s.c. injection. Both total hepatic porphyrin and LOOH levels were significantly (P<0.001) increased in the HCB-iron treated group as compared with the control group. Mice treated additionally with vitamin E had significant (P<0.001) lower levels as compared with the HCB-iron group. Similarly, the levels of 8-OHdG were significantly (P<0.001) increased above controls after HCB-iron treatment and this increase was reduced after co-treatment with vitamin E (P<0.02). The data support the hypothesis that the mechanism of hepatic porphyrinogenicity of HCB with iron overload is an oxidative free radical process.

Asunto(s)

Desoxiguanosina/metabolismo , Hexaclorobenceno/toxicidad , Hierro/toxicidad , Porfirias Hepáticas/prevención & control , Vitamina E/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Desoxiguanosina/análogos & derivados , Hierro/metabolismo , Peróxidos Lipídicos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Porfirias Hepáticas/inducido químicamente , Porfirias Hepáticas/metabolismo , Porfirinas/metabolismo , Vitamina E/metabolismo

RESUMEN

Chronic inflammation and fibrosis following quartz inhalation has been associated with persistent up-regulation of several "pro-inflammatory" genes, which are commonly regulated by nuclear factor kappa-B (NF-kappaB). Transcription of the NF-kappaB-inhibitor IkappaBalpha is also under NF-kappaB control, and its de novo synthesis is considered to comprise a negative feedback loop in transient inflammation. To investigate this mechanism in particle inflammation, we have studied IkappaBalpha degradation in A549 cells exposed to DQ12-quartz or TiO(2), in relation to the expression of IL-8. Although both quartz and TiO(2) were found to cause IkappaBalpha degradation, only quartz elicited a mild IkappaBalpha depletion, first appearing at 4 h. TiO(2) was found to cause a higher short-term increase in IkappaBalpha mRNA-expression compared to quartz, whereas the early enhancement of IL-8 expression and release was similar for both particles. Up-regulation of IL-8 expression was found to persist with quartz only. Cotreatment with PDTC and curcumin reduced particle-elicited IL-8 response, whereas cycloheximide caused enhancement of IL-8 mRNA expression in both the quartz- and TiO(2)-treated cells. Our results demonstrate that mineral dusts cause IkappaBalpha degradation, a transient increase in de novo synthesis of IkappaBalpha, and enhanced IL-8 expression in human pulmonary epithelial cells. While IkappaBalpha degradation and early IL-8 expression seem to be general particle phenomena, particle-specific characteristics impact on activation of IkappaBalpha gene transcription, apparently accounting for the different proinflammatory IL-8 responses seen with quartz and TiO(2) in the longer term. These observations may provide an explanation for the transient versus the persistent pulmonary inflammatory status and subsequent differences in pathogenic potency of TiO(2) and quartz.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Interleucina-8/metabolismo , Pulmón/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Prolina/análogos & derivados , Cuarzo/toxicidad , Western Blotting , Curcumina/farmacología , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Interleucina-8/genética , Pulmón/citología , Pulmón/metabolismo , Inhibidor NF-kappaB alfa , Prolina/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiocarbamatos/farmacología , Titanio/toxicidad , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

Ultrafine particles have been shown to induce pro-inflammatory effects both in vivo and in vitro. Increased expression of pro-inflammatory genes probably requires the activation of specific transcription factors such as nuclear factor kappa B (NF-kappaB) via a number of possible pathways including Ca2+ and reactive oxygen species. The fluorescent dye fura 2, was used to measure cytosolic Ca2+ in the human monocytic cell line, Monomac 6 on exposure to 66 microg x mL(-1) of either ultrafine carbon black (ufCB; diameter 14 nm), carbon black (CB; diameter 260 nm), quartz (diameter 1.45 microm), or medium alone. UfCB but not fine CB induced a 1.6-fold increase (p<0.01) in the resting cytosolic Ca2+ concentration of Monomac 6 cells. In addition ufCB induced a 2.6-fold increase (p<0.001) in the response to the endoplasmic reticulum Ca2+- adenosine triphosphatase (ATPase) inhibitor, thapsigargin, suggesting the Ca2+ release-activated Ca2+ current across the plasma membrane was enhanced. This response was inhibited by the removal of extracellular Ca2+ and by the Ca2+ channel blocker, verapamil. In addition, ufCB stimulated the entry of extracellular Mn2+. Finally, the antioxidants mannitol and nacystelin both inhibited the effects of ufCB on the response to thapsigargin. These data suggest that ultrafine carbon black particles stimulated an increase in cytosolic Ca2+, possibly through the entry of extracellular Ca2+ via Ca2+ channels in the plasma membrane. The particles may in part activate the opening of Ca2+ channels via a mechanism involving reactive oxygen species.

Asunto(s)

Calcio/metabolismo , Carbono , Monocitos/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Línea Celular , Citosol/metabolismo , Colorantes Fluorescentes , Fura-2 , Humanos , Técnicas In Vitro , Tamaño de la Partícula , Tapsigargina/farmacología

RESUMEN

Asbestos has been shown to stimulate the mitogen-activated protein kinase signaling cascade after autophosphoryiation of the epidermal growth factor recptor (EGF-R), an event important in regulating the response of cells to extracellular signals. In studies reported here, we have examined whether mineral fibers with known carcinogenicity can be discriminated from nonpathogenic fibers by their ability to upregulate expression of EGF-R protein in mesothelial cells. Crocidolite and erionite, two fibrous preparations known to induce mesothelioma, increased expression of EGF-R protein in a time- and dose-dependent manner, whereas milled (nonfibrous) crocidolite and chrysotile asbestos, two preparations with much less or no ability to induce mesothelioma, did not. Intense patterns of EGF-R protein expression were linked to mesothelial cells phagocytosing long fibers as observed by phase-contrast microscopy. To determine the importance of EGF-R expression in these cells, we assessed downstream signaling events in rat pleural mesothelial (RPM) cells by looking at the induction of activator protein-1 (AP-I), a transcription factor that controls the transition to S phase in the cell cycle, leading to cell proliferation. Crocidolite induced AP-I in RPM cells in a dose-dependent manner, and this induction of AP-I in RPM cells was inhibited by coincubation with tyrphostin AG 1478, a potent inhibitor of the EGF-R. To examine the mechanism of induction of EGF-R in RPM cells by asbestos, RPM cells were treated with crocidolite in the presence and absence of the antioxidant N-acetylcysteine (NAC). Reduced glutathione (GSH) was examined as a marker of oxidative stress and the expression of EGF-R protein was measured. Crocidolite asbestos caused a dose-dependent depletion of GSH in RPM cells, and the presence of NAC ameliorated the expression of EGF-R protein by crocidolite. Our data suggest that carcinogenic fibers induce EGF-R via a mechanism involving oxidative stress initiating cell signaling cascades in mesothelial cells leading to cell proliferation and carcinogenesis.

RESUMEN

Asbestos fibres have been shown to stimulate the mitogen-activated protein kinase signalling cascade in rat pleural mesothelial (RPM) cells after autophosphorylation of the epidermal growth factor receptor (EGFR). We examined if mineral fibres with known carcinogenicity can be discriminated from materials with less or no carcinogenicity by their ability to up-regulate expression of EGFR protein in RPM cells in vitro. Crocidolite and erionite, two fibrous preparations with marked potential to induce mesothelioma, were associated with increases in EGFR protein expression over sham controls, whereas chrysotile asbestos and milled (non-fibrous) crocidolite did not. Intense patterns of EGFR protein expression were linked to RPM cells phagocytosing long fibres. To determine the role of EGFR expression in these cells, we assessed cell proliferation using an antibody against proliferating cell nuclear antigen (PCNA) in combination with an antibody against EGFR. In these co-localization studies, cells showed intense staining for EGFR protein 24 h before being PCNA positive at 48 h. These results suggest that carcinogenic fibres induce EGFR and initiate cell signalling cascades in mesothelial cells, leading to cell proliferation and carcinogenesis.

Asunto(s)

Asbesto Crocidolita/toxicidad , Asbestos Serpentinas/toxicidad , Carcinógenos/toxicidad , Células Epiteliales/efectos de los fármacos , Receptores ErbB/genética , Pleura/citología , Zeolitas/toxicidad , Animales , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Masculino , Ratas , Ratas Endogámicas F344 , Regulación hacia Arriba

RESUMEN

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

Asunto(s)

Asbesto Crocidolita/farmacología , Liasas de Carbono-Oxígeno/metabolismo , Carcinógenos/farmacología , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Proteínas Nucleares/metabolismo , Pleura/enzimología , Animales , Northern Blotting , Liasas de Carbono-Oxígeno/genética , Células Cultivadas , Cartilla de ADN/química , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Técnica del Anticuerpo Fluorescente Indirecta , Microscopía Confocal , Mitocondrias/enzimología , Pleura/citología , Pleura/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344

RESUMEN

Respired ultrafine particles induce a greater inflammation in rat lungs than fine particles; we have hypothesized that this is due to their comparatively huge number and surface area for the production of free radicals. We tested this hypothesis by studying the effects of fine and ultrafine (uf) carbon black (CB) particles in comparison with quartz on A549 human type II alveolar epithelial cells, particularly with respect to the oxidative properties of these particles. Treatment with fine CB (diameter 260nm), and quartz (up to 0.78mug/mm(2)) for 24 hours significantly (P<0.05) decreased the A549 cells metabolic competence, as measured by the ability to reduce MTT to a formazan product. The inhibitory effects of uf CB only became significantly different (P<0.05) relative to the control at 48 hours, by which time the effects of fine CB and quartz were no longer significant. The inhibition of MTT reduction by uf CB was prevented by the hydroxyl radical scavenger mannitol (2mm). In addition, measurement of reactive oxygen species production using supercoiled plasmid DNA showed that uf CB exhibited significantly more free radical activity than fine CB (P<0.05). In the absence of serum, uf CB depleted reduced glutathione at 6 hours (P<0.008). In contrast, CB did not significantly alter reduced or oxidized glutathione. Hence, compared with fine CB, uf CB exhibited greater free radical activity, greater inhibition of the reduction of MTT at 48 hours (prevented by mannitol) and a depletion of reduced glutathione. These results suggest that uf CB induces a greater oxidative stress than fine CB, and that this may play a role in the toxicological effects of this ultrafine particle.

RESUMEN

Asbestos fibers cause persistent induction of the oxidative stress sensitive transcription factors nuclear factor kappa-B (NF-kappa B) and activator protein-1 (AP-1) in mammalian cells. These transcription factors play an important role in the regulation of cellular activity. Lipid peroxidation, mediated by reactive oxygen species, is thought to be a possible mechanism in the pathogenicity of asbestos fibers. These studies were designed to determine if crocidolite asbestos-induced lipid peroxidation plays a role in the mechanism of formation of NF-kappa B and AP-1. Treatment of a rat lung fibroblast cell line (RFL-6) with crocidolite asbestos in the presence and absence of the membrane antioxidant vitamin E decreased the levels of crocidolite-induced AP-1 and NF-kappa B to background levels. Preincubation of RFL-6 cells with 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, prior to exposure to crocidolite, abrogated crocidolite-induced NF-kappa B DNA-binding activity to background levels. Coincubation with indomethacin, a cyclooxygenase inhibitor, had no effect on NF-kappa B DNA-binding activity induced by crocidolite. However, nordihydroguaiaretic acid, a lipoxygenase inhibitor, decreased levels of NF-kappa B to background levels. This would suggest that lipoxygenase metabolites of arachidonic acid, produced following lipid peroxidation, are involved in the cellular signalling events to NF-kappa B transcription factor induction by asbestos.

Asunto(s)

Asbesto Crocidolita/toxicidad , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Peroxidación de Lípido/fisiología , FN-kappa B/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Vitamina E/farmacología , Animales , Ácido Araquidónico/metabolismo , Línea Celular , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , ADN/biosíntesis , Peroxidación de Lípido/efectos de los fármacos , Inhibidores de la Lipooxigenasa/farmacología , Ratas

RESUMEN

A Criteria Document for hexavalent chromium [Cr(VI)], currently under preparation at the Institute of Occupational Health, University of Birmingham, is intended for use in setting an occupational exposure limit (OEL) for Cr(VI) in the European Union (EU). The requirement for establishing OELs, specifically known as indicative limit values, in the EU is set out in Council Directive 80/1107/EEC, amended by Council Directive 88/642/EEC. To facilitate this procedure the Commission has set up a Scientific Committee for Occupational Exposure Limits to Chemical Agents. The Committee, which is composed of independent scientific experts from member states, is responsible for reviewing available scientific data. A Criteria Document forms the basis of the scientific data considered during this process and contains an up-to-date, critical evaluation of available information which is relevant to setting an exposure limit. After consideration of the scientific data for a particular substance, the Committee advises the Commission on setting a health-based OEL. Consideration of other questions such as technical matters and socioeconomic issues occurs during later stages of the procedure, before an OEL is finally adopted. The procedure allows for consultation with interested parties. The Criteria Document for Cr(VI) contains sections on substance identification, chemical and physical properties, production and use data, recent data on occupational exposure, current methods for measurement and analysis, and toxicology. The toxicology section contains a critical evaluation of both human and animal data and forms a major part of the document. This section enables identification of critical health effects associated with exposure to Cr(VI) and consideration of dose-response relationships and provides the basis for any risk assessment and recommendation for an OEL.

Asunto(s)

Compuestos de Cromo/efectos adversos , Exposición Profesional/legislación & jurisprudencia , Carcinógenos/efectos adversos , Carcinógenos/análisis , Compuestos de Cromo/análisis , Unión Europea , Humanos , Exposición Profesional/efectos adversos , Exposición Profesional/análisis

RESUMEN

Parental smoking data have been reabstracted from the interview records of the Oxford Survey of Childhood Cancers (deaths from 1971 to 1976). Reported smoking habits for the parents of 2587 children who died with cancer were compared with similar information for the parents of 2587 healthy controls (matched pairs analysis). Maternal daily consumption of cigarettes and paternal use of pipes or cigars were unimportant, but there was a statistically significant positive trend between paternal daily consumption of cigarettes and the risk of childhood cancer (P < 0.001). This association could not be explained by maternal smoking, social class, parental ages at the birth of the survey child, sibship position or obstetric radiography. Relations between maternal consumption of cigarettes and birth weights suggested that (maternal) smoking data were equally reliable for case and control subjects. About 14% of all childhood cancers in this series could be attributable to paternal smoking. These data were combined with smoking data from two previously published reports from the Oxford Survey (deaths from 1953 to 1955, deaths from 1977 to 1981) to obtain further information on risks for different types of cancer and different ages at onset of disease. Paternal cigarette smoking emerged as a potential risk factor both for the generality of childhood cancer and for all ages at onset.

Asunto(s)

Neoplasias/etiología , Padres , Fumar/efectos adversos , Contaminación por Humo de Tabaco/efectos adversos , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Neoplasias/epidemiología , Factores de Riesgo

RESUMEN

Epidemiological studies of workers exposed to fumes in the iron and steel foundry industry have consistently demonstrated an increased relative risk of lung cancer of approximately 1.4. Foundry fume is a complex mixture of gases and fine particles generated during the casting process when molten metal is poured into sand moulds bound together with organic binders. The chemical composition of fume varies according to foundry process and, specifically, binder composition. Previous in vitro studies have demonstrated that some fumes have mutagenic activity and that this varies with fume type. The current study has examined the potential carcinogenicity of three fumes in a 2-yr in vivo rodent bioassay using an intrabronchial pellet implantation technique. The toxicity and genotoxicity of the fumes were tested concurrently in a number of in vitro assays including those identifying mutagenicity, unscheduled DNA synthesis, free radical DNA damage and micronucleus induction. The rodent bioassay failed to demonstrate a carcinogenic response, although an increase in preneoplastic lesions was seen in all fume-treated groups relative to controls. When tested in vitro, the fumes were positive in many assays and activity correlated with the polycyclic aromatic hydrocarbon content of the fumes. The employment of a combination of in vitro assays for different genotoxic endpoints, such as those presented in the current study, provides information useful for the overall assessment of carcinogenicity of complex mixtures such as foundry fume.

Asunto(s)

Bronquios/efectos de los fármacos , Carcinógenos/toxicidad , ADN/efectos de los fármacos , Sustancias Peligrosas/toxicidad , Metalurgia , Hidrocarburos Policíclicos Aromáticos/toxicidad , Tráquea/efectos de los fármacos , Animales , Bronquios/patología , Línea Celular , Células Cultivadas , ADN/biosíntesis , Femenino , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/epidemiología , Masculino , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Exposición Profesional , Hidrocarburos Policíclicos Aromáticos/análisis , Conejos , Ratas , Ratas Wistar , Acero , Tráquea/patología

RESUMEN

Treatment of isolated DNA with crocidolite and man-made vitreous fibre-21 (MMVF-21) significantly increased the concentration of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA above background levels and co-treatment with glutathione (GSH) eliminated this effect. Crocidolite, MMVF-21 and chrysotile fibres increased the number of revertants in Salmonella typhimurium TA100 and GSH-deficient strains, TA100/NG-54 and TA100/NG-57, over background levels. This increase was small in TA100 but was greater in the GSH-deficient strains. When these bacterial strains were further depleted of GSH by co-culture with buthionine sulfoximine, all fibres tested caused a significant increase in the number of revertants over the parent strains. Pre-treatment with the GSH precursor N-acetyl-L-cysteine reduced the number of revertants to below that of the parent strain. Previous studies have shown a mechanistic role for iron-catalyzed production of oxygen radicals in the mutagenicity of fibres and this study suggests a protective role for GSH against such oxidative damage possibly by acting as a radical scavenger.

Asunto(s)

Asbesto Crocidolita , Aductos de ADN/metabolismo , ADN Bacteriano/metabolismo , Desoxiguanosina/análogos & derivados , Glutatión/metabolismo , Fibras Minerales , Mutágenos , 8-Hidroxi-2'-Desoxicoguanosina , Desoxiguanosina/metabolismo , Pruebas de Mutagenicidad , Oxidación-Reducción , Salmonella typhimurium

RESUMEN

Although well-established as carcinogens, the way in which chromium (VI) compounds exert their carcinogenic, mutagenic, and DNA-damaging potential remains obscure. It is clear that inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH). The present study is intended to clarify if Fenton mechanisms are likely to be important in the formation of DNA lesions by chromium(VI) in combination with GSH. In buffer solutions which were treated to remove Fenton-active metal ions as well as in those not further purified, chromate and GSH induced similar numbers of single-strand breaks (SSB) in isolated PM2 DNA. Molecular oxygen was found to be essential for the formation of SSB, but chromium(V) species arising from chromate/GSH, unless activated by oxygen, appeared to be unreactive toward DNA. Upon addition of Mn(II) to solutions of chromium(VI) and GSH a diminution of Mn(II) ESR signals was observed, good evidence for the presence of chromium(IV) species. Using gas chromatography/mass spectrometry in selective ion-monitoring mode and high-performance liquid chromatography with electrochemical detection, we were able to show that Cr(VI)/GSH failed to induce base modifications typical of hydroxyl radical attack on DNA. Experimental conditions which readily induced SSB gave rise to the formation of chromium-DNA adducts, clearly demonstrating that the generation of these two DNA lesions is not mutually exclusive. We conclude that models which ascribe the induction of chromium-DNA adducts to chromium(V) and the generation of oxidative DNA damage including SSB to hydrogen peroxide are oversimplistic. It is not necessary to invoke a mechanism requiring the presence of added hydrogen peroxide to account for the ability of Cr(VI)/GSH to cause SSB. Our findings suggest that the combination of GSH, molecular oxygen, and chromium(VI) can damage DNA via non-Fenton pathways.

Asunto(s)

Carcinógenos Ambientales/metabolismo , Cromo/metabolismo , Daño del ADN , Glutatión/metabolismo , Oxígeno/metabolismo , Radical Hidroxilo , Hierro/metabolismo , Oxidación-Reducción

RESUMEN

Certain end-products of lipid peroxidation bind to DNA forming a fluorescent chromophore. Incubation of both Salmonella typhimurium TA104 and a rat lung fibroblast cell line, RFL-6, with various types of mineral fibre resulted in a time- and dose-dependent increase in DNA fluorescence. The increase in DNA fluorescence was shown to be directly related to the amount of iron that could be mobilized from the fibre surface using in vitro studies in the absence of cells or bacteria. Crocidolite and man-made vitreous fibre-21 (MMVF-21) mobilized significant quantities of iron and were significantly more active than chrysotile and refactory ceramic fibre-1 (RCF-1). Fibre-induced malondialdehyde-DNA adduct formation, the fluorescent product, was increased by incubating cells with buthionine sulfoximine and ameliorated by co-treatment with N-acetylcysteine, indicating a protective role for glutathione. Similarly, vitamin E was also shown to inhibit DNA adduct formation. These results suggest that mineral fibre-induced lipid peroxidation produced genotoxic products which can diffuse into nucleus and interact with cellular DNA. In conclusion, fibre-induced lipid peroxidation may be a possible mechanism in the genotoxic action of fibrous materials.

Asunto(s)

Asbesto Crocidolita/toxicidad , Asbestos Serpentinas/toxicidad , Carcinógenos/toxicidad , Aductos de ADN/biosíntesis , Fibroblastos/metabolismo , Peroxidación de Lípido , Salmonella typhimurium/metabolismo , Acetilcisteína/farmacología , Animales , Antídotos/farmacología , Butionina Sulfoximina , ADN/metabolismo , Deferoxamina/farmacología , Inhibidores Enzimáticos/farmacología , Ferrozina/farmacología , Vidrio , Glutatión/metabolismo , Pulmón/citología , Malondialdehído/metabolismo , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Fibras Minerales/toxicidad , Ratas , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo

RESUMEN

The potential importance of prostaglandin H synthase (PGHS) in the genotoxicity of fecapentaene-12 (fec-12) has been indicated by the finding that non-steroidal anti-inflammatory agents (NSAIDs) block the induction of oxidative DNA base damage by fec-12 in HeLa cells. To further investigate the role of PGHS in the metabolic 'activation' and genotoxicity of fec-12, we have measured: (i) oxygen uptake by fec-12 with purified preparations of PGHS; and (ii) the induction of DNA single-strand breaks (SSBs) in HeLa cells exposed to fec-12 in the absence or presence of PGHS inhibitors. Oxygen uptake occurred spontaneously with fec-12 alone but was stimulated 3-fold by the presence of PGHS. The potentiation of fec-12 oxygenation by PGHS was independent of arachidonate and inhibited 45% by indomethacin (INDO). Methylphenylsulphide (MPSI), a reducing substrate expected to compete with fec-12 in peroxidase-dependent co-oxidation reactions, also inhibited PGHS-mediated oxygen uptake with fec-12 by 55%. These results, together with the observation that the inhibitory effects of these agents in combination were additive, suggest that both the cyclooxygenase and peroxidase components of PGHS are involved in the oxidation of fec-12. INDO and MPSI also blocked the induction of DNA SSBs by fec-12 in HeLa cells, indicating that both components of PGHS are also involved in potentiating the genotoxicity of fec-12. It is proposed that this occurs in two ways: firstly, by formation of highly reactive fec-12 hydroperoxides which would generate oxygen radicals through Fenton-like reactions, and secondly, by the generation of oxygen radicals through peroxidase-mediated co-oxidation of fec-12.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Daño del ADN , Mutágenos/toxicidad , Polienos/toxicidad , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sinergismo Farmacológico , Células HeLa , Humanos , Indometacina/farmacología , Cinética , Oxidación-Reducción , Consumo de Oxígeno , Peroxidasas/antagonistas & inhibidores , Sulfuros/farmacología

RESUMEN

Exposure of human white blood cells to UICC crocidolite asbestos in vitro resulted in the formation of DNA strand breakage in a dose-dependent manner up to a fibre concentration of 100 micrograms/ml. Subsequent incubations with the iron chelator desferrioxamine or the intracellular Ca2+ chelator Quin-2 prevented DNA strand break formation above control incubations. Addition of aurintricarboxylic acid, an endonuclease inhibitor, similarly abolished crocidolite-induced DNA strand breaks in these cells. These results suggest that crocidolite-derived hydroxyl radicals do not directly induce DNA strand breakage in mammalian white blood cells. In order to assess Ca2+ mobilisation from intracellular stores in control and crocidolite-treated cells, the fullness of these stores was measured by treating with thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. On addition of thapsigargin to fura-2AM-loaded cells treated with crocidolite we demonstrated that the endoplasmic reticulum stores had been depleted as no further Ca2+ was released, unlike control cells. We suggest that strand breakage is caused by a complex set of events involving oxygen free radicals that may disturb intracellular Ca2+ homoeostasis and the breaks are produced by secondary reactions, involving Ca(2+)-mediated enzymes.

Asunto(s)

Aminoquinolinas/farmacología , Asbesto Crocidolita/toxicidad , Calcio/metabolismo , Quelantes/farmacología , Estrés Oxidativo , Antimutagênicos/farmacología , Ácido Aurintricarboxílico/farmacología , Calcio/agonistas , Daño del ADN , Deferoxamina/farmacología , Radicales Libres/metabolismo , Humanos , Leucocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

OBJECTIVES: Molecular epidemiological techniques, capable of detecting damage to DNA, were used to see if such damage occurred in the lymphocytes of a group of workers exposed to chromium. The two aims of this pilot study were to see if these new techniques might make useful biological monitoring tools for workers exposed to chromium and also, to help assess whether the current occupational exposure limit for chromium (VI) was sufficiently protective in this specific working situation. METHODS: Volunteer groups of 10 workers exposed to chromium and 10 non-exposed workers provided urine and blood samples towards the end of the working week. Chromium concentrations were measured in whole blood, plasma, lymphocytes, and urine. Lymphocytes were used to examine two forms of DNA damage in the two groups; these were the level of DNA strand breakage and, the production of 8-hydroxydeoxyguanosine. RESULTS: Chromium concentration in whole blood, plasma, and urine of workers exposed to chromium was significantly raised (P < 0.01) compared with non-exposed controls, but in isolated lymphocytes, there was only a modest but significant (P < 0.05) increase in chromium in the group exposed to chromium. There was no difference in the levels of DNA strand breaks or 8-hydroxydeoxyguanosine between the groups. Air monitoring for chromium was not undertaken but current levels for the group exposed to chromium were reported to be around 0.01 mg/m3, which is 20% of the current United Kingdom occupational exposure limit. CONCLUSIONS: We were unable to detect any damage in lymphocytic DNA due to exposure to chromium. This may have been due to the low chromium exposure (< 20% of the United Kingdom occupational exposure limit), the ability of plasma to detoxify chromium (VI) to chromium (III) before it reached the lymphocytes, or perhaps the insensitivity of the molecular techniques used. It is now important to test these and other such techniques on groups exposed to levels closer to the United Kingdom occupational exposure limit.

Asunto(s)

Industria Química , Cromo/administración & dosificación , Daño del ADN , Monitoreo del Ambiente/métodos , Exposición Profesional , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Cromo/efectos adversos , Cromo/análisis , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Femenino , Humanos , Linfocitos/química , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Fumar/sangre

RESUMEN

Treatment of isolated DNA with crocidolite asbestos significantly increased the concentration of 8-hydroxydeoxyguanosine (8-OHdG) above background. Furthermore, incubating DNA with H2O2 and crocidolite potentiated the formation of 8-OHdG above levels observed with crocidolite alone. In the presence of desferrioxamine, desferrioxamine and ferrozine, dimethylsulphoxide (DMSO) or o-phenanthroline, crocidolite-induced DNA oxidation was reduced by 36, 73, 74 and 70% respectively. Crocidolite, but not chrysotile asbestos, enhanced background revertants in Salmonella typhimurium TA102, at sub-cytotoxic concentrations in a dose-dependent manner. The mutagenic effects of crocidolite were quite small and this indicates that crocidolite was a weak mutagen in this study. The number of revertants was reduced to the spontaneous rate for this strain after the fibres had been pretreated with desferrioxamine before assaying for genotoxicity in this oxygen radical-sensitive strain. These results help to explain a mechanistic role for iron in crocidolite-induced DNA oxidation and mutagenicity in TA102.

Asunto(s)

Asbesto Crocidolita/toxicidad , ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Hierro/fisiología , Mutación , 8-Hidroxi-2'-Desoxicoguanosina , ADN/metabolismo , Deferoxamina/farmacología , Desoxiguanosina/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética

RESUMEN

The genotoxic/mutagenic mechanism(s) of action of fecapentaene-12 (fec-12) is complex but there is evidence to suggest that the generation of active oxygen species (AOS) may be involved. This has been assessed by measuring the formation of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA and HeLa cells exposed in vitro to fec-12. The possibility that fec-12 may form AOS via peroxidative 'activation' by prostaglandin H synthase (PHS) has been investigated by measuring 8-OHdG in HeLa cells exposed to fec-12 in the absence or presence of PHS inhibitors. The role of iron as a catalyst in this pathway has also been investigated. A 4-fold increase in the level of 8-OHdG in isolated DNA was seen after exposure to fec-12 (1 mM) alone. This increase was enhanced synergistically by ferrous iron. Fec-12 exposure of HeLa cells at 50 and 100 microM induced 2- and 3-fold increases (P < 0.001) respectively in the level of 8-OHdG in cellular DNA. No increase was seen at 10 microM fec-12. The PHS inhibitors indomethacin and acetylsalicylate blocked the formation of 8-OHdG induced by fec-12 (50 microM) but did not inhibit the formation of 8-OHdG in these cells after exposure to H2O2 and Fe2+. Addition of the iron chelating agent o-phenanthroline to cells prior to fec-12 exposure blocked the increase in 8-OHdG induced by fec-12 (50 microM). Addition of the radical scavenging agent DMSO (10%) to cells prior to fec-12 exposure reduced the level of 8-OHdG to within 10% of control. Specific inhibition of fec-12 induced 8-OHdG formation in HeLa cells by PHS inhibitors suggests that this enzyme may be involved in 'activating' fec-12 to form AOS in cells. Inhibition of fec-12 induced 8-OHdG formation in cells by o-phenanthroline suggests a role for intracellular iron as a catalyst in this process.

Asunto(s)

ADN/metabolismo , Desoxiguanosina/análogos & derivados , Polienos/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Inhibidores de la Ciclooxigenasa/farmacología , Desoxiguanosina/biosíntesis , Células HeLa/metabolismo , Humanos , Indometacina/farmacología , Fenantrolinas/farmacología , Polienos/metabolismo , Salicilatos/farmacología