RESUMO
Mainstream cigarette smoke (MSS) from 12 US cigarette brands and two reference cigarettes was evaluated to determine concentrations of dioxins (i.e., polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs)). The study included three 'tar' ranges based on Federal Trade Commission (FTC) determination: Low Yield (LY) < or = 5.5, Medium Yield (MY) 9.6-12.2, and High Yield (HY)> or = 14.5 mg/cig. Of the brands studied, the HY cigarettes yielded the greatest mean concentrations of 2005 World Health Organization Toxic Equivalents (WHO-TEQs) on a per cigarette basis. WHO-TEQ levels in LY cigarettes were significantly lower than for HY cigarettes (p=0.039) on a yield per cigarette basis and WHO-TEQ concentrations correlated with 'tar' yield (r=0.73, p=0.007), as did concentration on a WHO-TEQ per body mass per day basis (r=0.73, p=0.007). However, a statistically significant relationship was not observed between 'tar' yield levels and WHO-TEQ concentrations on a per mg Total Particulate Matter (TPM) basis. Concentrations for all brands tested ranged from 0.44 to 3.88 fg WHO-TEQ/mg TPM. Maximum daily exposure estimates calculated from this range (0.004-0.074 pg WHO-TEQ/kg bw/day) are below the current WHO Tolerable Daily Intake range of 1-5 pg/kg bw/day.
Assuntos
Dioxinas/análise , Poluentes Ambientais/análise , Nicotiana/química , Fumaça/análise , Interpretação Estatística de Dados , Filtração , Material Particulado/análise , Bifenilos Policlorados/análise , Dibenzodioxinas Policloradas/análogos & derivados , Dibenzodioxinas Policloradas/análise , Padrões de Referência , Medição de Risco , Estados UnidosRESUMO
This study demonstrates that in vitro exposure of adult rat alveolar epithelial cells to CdCl(2) decreases DNA binding activity of specificity protein 1 (Sp1), a zinc-finger transcription factor known to play a key role in eukaryotic gene expression, maintenance of homeostasis, cell cycle control, terminal differentiation, and apoptosis. Suppression of Sp1 function, as assessed by electrophoretic mobility shift assays (EMSAs), is dependent upon cadmium (Cd) dose and duration of exposure. A 45% decrease of Sp1 activity occurs as early as 30 min after Cd addition. By 2 h, Sp1 activity is reduced even further with no loss of cell viability, suggesting that Sp1 inactivation precedes cell death. If Cd is removed from cultures during these early periods of exposure, inhibition of Sp1 binding activity is reversed. Sp1 inactivation does not appear to be a generalized, non-selective response to Cd as other transcription factors are up-regulated under the same conditions. Phosphorylation is involved in Sp1 down-regulation, as evidenced by the finding that alkaline phosphatase treatment of nuclear extracts from cells exposed to Cd for 2 h helps restore Sp1 binding activity. A broad spectrum Protein Kinase C (PKC) inhibitor, GF109203X, substantially reduces the Cd-mediated effect on Sp1 suggesting that a member of the PKC family is required for Sp1 phosphorylation. More prolonged Cd exposure promotes Sp1 degradation with the appearance of cleavage products (40 and 50 kDa), as detected by Western blotting. Changes in the integrity of the Sp1 protein are accompanied by a corresponding decline in cell survival. Cd-induced cell death is substantially attenuated if cells are pretreated with antagonists of PKC activity which implies that a PKC isoform is also a participant in this process.
Assuntos
Cádmio/farmacologia , Células Epiteliais/metabolismo , Alvéolos Pulmonares/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Radioisótopos de Cádmio , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Meios de Cultura , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Oxirredução , Fosforilação , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Tiazóis , Fatores de TempoRESUMO
This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.
Assuntos
Cádmio/toxicidade , Dano ao DNA , Reparo do DNA , Peróxido de Hidrogênio/farmacologia , Metalotioneína/metabolismo , N-Glicosil Hidrolases/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Células Cultivadas , Ensaio Cometa , DNA-Formamidopirimidina Glicosilase , Interações Medicamentosas , Endonucleases/metabolismo , Cinética , Metalotioneína/isolamento & purificação , N-Glicosil Hidrolases/antagonistas & inibidores , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/enzimologia , RatosRESUMO
This review article discusses the major cellular and molecular responses characterizing pulmonary adaptation to cadmium (Cd) that may ultimately contribute to Cd carcinogenesis. Hallmarks of Cd adaptation include hyperplasia and hypertrophy of type II alveolar epithelial stem cells, an inflammatory response involving polymorphonuclear leukocytes, and the increased gene and protein expression of several resistance factors. The most prominent biochemical change is associated with Cd-induced up-regulation of metallothionein, a cysteine-rich, metal-binding protein that sequesters Cd and also possesses considerable free radical scavenging ability. Increased levels of glutathione (GSH) and induction of enzymes involved with both the synthesis of GSH (gamma-glutamylcysteine synthetase regulatory and catalytic subunits) and its metabolism (GSH S-transferases) also constitute important components of the pulmonary adaptive response. Enhancement of several important cellular defense systems in response to Cd exposure may, at first, appear to be beneficial. However, recent evidence suggests that the Cd-adaptive phenotype could have deleterious consequences and may represent a double-edged sword. It has been discovered that Cd-adapted alveolar epithelial cells have a reduced ability to repair DNA damage due, in part, to the inhibition of two base excision repair enzymes (8-oxoguanine-DNA glycosylase and endonuclease III). Cells with genetic aberrations resulting from unrepaired DNA lesions would normally be removed from the lung by apoptosis. However, another study has demonstrated that apoptotic cell death, following an oxidant challenge, is significantly attenuated in Cd-adapted cells compared to non-adapted counterparts. Suppressed apoptosis could leave pre-neoplastic or neoplastic cells alive, favor their clonal expansion, and ultimately promote tumor development. The presence of superior antioxidant defenses would also be expected to increase the resistance of these tumors to chemotherapeutic agents.
Assuntos
Adaptação Fisiológica/fisiologia , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Administração por Inalação , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cádmio/administração & dosagem , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Glutationa/metabolismo , Técnicas In Vitro , Metalotioneína/metabolismo , Modelos Animais , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Regulação para CimaRESUMO
Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.
Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Peróxido de Hidrogênio/antagonistas & inibidores , Neoplasias Pulmonares/induzido quimicamente , Oxidantes/antagonistas & inibidores , Alvéolos Pulmonares/efeitos dos fármacos , Adaptação Biológica , Cádmio/farmacologia , Catálise , Núcleo Celular/efeitos dos fármacos , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Glutationa/metabolismo , Glutationa S-Transferase pi , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Marcação In Situ das Extremidades Cortadas , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Metalotioneína/biossíntese , Metalotioneína/genética , Oxidantes/farmacologia , Isoformas de Proteínas , Alvéolos Pulmonares/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.
Assuntos
DNA/genética , Estresse Oxidativo/fisiologia , Sêmen/fisiologia , Espermatozoides/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Dano ao DNA , Fragmentação do DNA , DNA Nucleotidilexotransferase/metabolismo , Humanos , Peroxidação de Lipídeos , MasculinoRESUMO
Exposure of rat alveolar epithelial cells to 10 micromol/L CdCl2 causes time-dependent increases in steady-state mRNA levels of the gamma-glutamylcysteine synthetase catalytic (heavy) subunit (gamma-GCS) and of glutathione S-transferase isoforms (GST-alpha and GST-pi). The expression of gamma-GCS was significantly increased as early as 2 h after addition of cadmium. Maximal induction of gamma-GCS mRNA (approximately 4-fold), at 8 h, was subsequently followed by increases in gamma-GCS activity/protein and glutathione (GSH) levels. Maximal elevations in GST-pi (approximately 2-fold) and GST-alpha (approximately 10-fold) transcripts, at 8 and 24 h, respectively, were also accompanied by enhanced GST activity. Cadmium-induced oxidative stress, assessed by alterations in GSH homeostasis and an accelerated rate of intracellular oxidant production, could constitute early events in the signal transduction pathway mediating these responses. The dimeric transcription factor, activator protein-1 (AP-1), may also play a regulatory role in this process. This association is suggested by transcriptional activation of the immediate-early response genes, c-fos and c-jun, within 15 min after exposure to cadmium and by the enhancement of AP-1 DNA binding activity, involving a c-Jun protein complex, which is maximally induced (approximately 4-fold) by 2 h. These molecular changes likely function together to protect alveolar epithelial cells against cadmium toxicity.
Assuntos
Cádmio/farmacologia , Glutamato-Cisteína Ligase/genética , Glutationa Transferase/genética , Isoenzimas/genética , Estresse Oxidativo/fisiologia , Alvéolos Pulmonares/citologia , Fator de Transcrição AP-1/metabolismo , Animais , Northern Blotting , Domínio Catalítico , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Precoces/fisiologia , Glutamato-Cisteína Ligase/metabolismo , Glutationa S-Transferase pi , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , RNA Mensageiro/análise , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The human epididymis provides an optimal environment for the storage and maturation of spermatozoa. However, the ability of the epididymis to protect spermatozoa from oxidative attack whilst stored at this site, through the local actions of antioxidants, has not thus far been well studied. This study assessed the contribution of the epididymis to seminal plasma antioxidant activity, by comparing the semen of normozoospermic and vasectomized men. Total seminal plasma antioxidant activity was measured, as were concentrations of urate, ascorbate and thiols, antioxidants that are abundant in human semen. Thiobarbituric acid reactive species (TBARS) were measured to indicate lipid peroxidation. Total antioxidant activity and thiol content were significantly lower (P < 0.05) in the plasma from vasectomized men compared with that of normozoospermic donors. Ascorbate and urate were found at similar concentrations in the plasma of both groups. The concentration of TBARS was significantly higher (P < 0.001) in the semen from vasectomized individuals compared with the normozoospermic group. The results indicate that the epididymis contributes to the antioxidant capacity of seminal plasma and possesses region-specific antioxidant activity, which may potentially protect spermatozoa from oxidative attack during storage at this site.
Assuntos
Antioxidantes/metabolismo , Epididimo/metabolismo , Ácido Ascórbico/metabolismo , Humanos , Masculino , Compostos de Sulfidrila/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Ácido Úrico/metabolismoRESUMO
Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.
Assuntos
Núcleo Celular/patologia , Epididimo/citologia , Espermatozoides/patologia , Cromatina/química , Cromatina/patologia , DNA/química , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Desnaturação de Ácido Nucleico , Sêmen , VasectomiaRESUMO
Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.