RESUMEN
ß-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on ß-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with ß-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of ß-carotene. Their generation originates in the polyene structure of ß-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs). Due to their instability and hydrophobicity, the handling of standards and real samples containing ß-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.
Asunto(s)
Técnicas de Química Analítica , Depuradores de Radicales Libres/análisis , Oxidantes , beta Caroteno/análisis , Animales , Bioensayo , Calibración , Células Cultivadas , Técnicas de Química Analítica/normas , Estabilidad de Medicamentos , Depuradores de Radicales Libres/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Oxidantes/química , Oxidación-Reducción , Estándares de Referencia , Solubilidad , Relación Estructura-Actividad , beta Caroteno/químicaRESUMEN
A validated ultrahigh-performance liquid chromatography method using 1.7 µm core-shell particles is presented for the identification and quantification of ß-carotene (BC) and related cleavage products (CPs) in primary cell culture media. Besides BC, apo-4'-, apo-8'-, apo-10'-, and apo-12'-carotenals, as well as 5,6-epoxy-ß-carotene, were selected as target analytes. Detection was performed via an 80-Hz diode array detector and an electrospray ionization-linear quadrupole ion trap-Orbitrap XL mass spectrometer, both hyphenated in series. Total analysis time was below 6 min with peak widths <12 s. Addition of trifluoroacetic acid and tetrahydrofuran to the mobile phase allowed for the mass spectrometric detection of BC and related CPs and reduced peak tailing due to improved solubility of hydrophobic analytes. Intra-day and inter-day precision for UV and mass spectrometric detection were ≤1.5 % for retention times and ≤5.1 % for peak areas. Instrumental linearity was confirmed by Mandel's fitting test between 0.25 (or 1.00 µg/mL) and 5.00 µg/mL for UV detection. The higher sensitivity of mass spectrometric detection allowed for the coverage of three concentration domains between 0.025 and 5.00 µg/mL in linearity testing. Homoscedasticity was confirmed between 0.10 and 5.00 µg/mL for Orbitrap XL MS. The limits of quantification were between 52.6 and 889.4 ng/mL for UV detection and between 19.3 and 102.4 ng/L for mass spectrometric detection. Offline solid-phase extraction from culture media fortified with BC and CPs provided intra- and inter-day recoveries between 65.8 and 102.4 % with coefficients of variation ≤6.2 %. Primary rat hepatocyte cultures treated with BC and subjected to different oxidative stress conditions contained 5,6-epoxy-BC and apo-4'-carotenal besides residual BC. Apparently, 5,6-epoxy-BC was formed in the medium via autoxidation of BC by ambient oxygen.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , beta Caroteno/química , beta Caroteno/aislamiento & purificación , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión/instrumentación , Femenino , Hepatocitos/química , Estructura Molecular , Ratas , Ratas Endogámicas F344RESUMEN
In rats, N-nitrosodiethylamine (NDEA) induces tumors mainly in the liver. This could be because various enzymes are responsible for the metabolic activation of NDEA, besides the hepatic NDEA metabolizing enzyme, CYP2E1. We examined NDEA genotoxicity and cytotoxicity in primary cultures of female rat hepatocytes; we also looked at how it affected CYP mRNA expression. Single incubation with 0.9% NaCl resulted in a mean of 0.2% apoptotic cells, which doubled with 105 µg NDEA/mL. The frequency of necrosis with NDEA treatment was also doubled. Besides the cytotoxic effects, there was also a 4-fold decrease in mitotic index and a 3-fold decrease in the percentage of cells with micronuclei. A significant increase in micronucleus cells when hepatocytes were incubated with 2.1 µg NDEA/mL suggests that DNA repair was inactive. The chromosomal aberration evaluation revealed a discrete dose-response curve. Treatment with NDEA induced increases in CYP mRNA: CYP2B2 (1.8 times) and CYP2E1 (1.6 times) with non-cytotoxic NDEA concentrations (0.21-21 µg/mL). CYP2B1 mRNA levels decreased at 0.21 µg NDEA/mL (2.5-fold), while CYP4A3 mRNA decreased 1.3-fold. NDEA treatment at 2.1 µg/ mL induced a 1.9-fold increase in CYP3A1 mRNA. Understanding the cumulative effects in target cells during precarcinogenesis is crucial to understanding the mode of action of potential carcinogens and in order to develop comprehensive chemical toxicity profiles.
Asunto(s)
Alquilantes/farmacología , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Citocromo P-450 CYP2E1/biosíntesis , Daño del ADN/efectos de los fármacos , Dietilnitrosamina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatocitos/enzimología , Esteroide Hidroxilasas/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Hepatocitos/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Necrosis/enzimología , Necrosis/patología , ARN Mensajero , Ratas , Ratas Endogámicas F344RESUMEN
A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of ß-carotene in cell culture media has been developed. Target CPs comprised ß-ionone (ß-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 µg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for ß-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method.
Asunto(s)
Células Epiteliales Alveolares/química , Hepatocitos/química , Extracción en Fase Sólida , beta Caroteno/análisis , Células Epiteliales Alveolares/citología , Animales , Células Cultivadas , Cromatografía de Gases y Espectrometría de Masas , Hepatocitos/citología , Estructura Molecular , Ratas , EstereoisomerismoRESUMEN
Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as 'second messengers' of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.
Asunto(s)
Aldehídos/metabolismo , Peroxidación de Lípido/fisiología , Estrés Oxidativo/fisiología , Aldehídos/química , Animales , Biomarcadores/metabolismo , HumanosRESUMEN
AIM OF THE STUDY: Eryngium creticum, Nigella sativa, and Teucrium polium have been traditionally used for the treatment of inflammations, liver disorders, and arthritis. Various studies on these plants revealed anti-inflammatory, hepatoprotective and antimutagenic activities. Previous results of our research group, however, indicate that aqueous extracts prepared as for the traditional use (tea) have neither cytoprotective nor antimutagenic activity. Instead, there is evidence for a mutagenic potential. Since the described antimutagenic activity may not be present in effective amounts in the aqueous extracts this study focuses on ethanolic extracts. MATERIALS AND METHODS: Ethanolic extracts of the three plant species were prepared and tested against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a directly acting mutagen. Since it cannot be excluded that the active constituents of the plant extracts require biotransformation or induce metabolic enzymes, causing antimutagenic or detoxifying effects, primary cultures of rat hepatocytes were used for this study. Plant ethanolic extracts were applied along with MNNG in three protocols: pre-treatment, combined treatment and post-treatment. RESULTS AND CONCLUSIONS: The results of this investigation clearly indicate an inhibitory effect of the plant extracts on MNNG mutagenicity, while the extracts had no effect on cytotoxicity indicators such as necrosis and apoptosis. The effects obtained can be attributed to a direct antimutagenic activity and an increased recovery at the chromosomal level. In order to identify the responsible compounds extracts will in a next step have to be fractionated, tested and chemically analyzed.
Asunto(s)
Antimutagênicos/farmacología , Árabes , Etanol/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales/fisiología , Animales , Antimutagênicos/aislamiento & purificación , Células Cultivadas , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Medio Oriente , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales/química , Ratas , Ratas Endogámicas F344RESUMEN
Nigella sativa has been traditionally used for the treatment of inflammations, liver disorders, and arthritis. Experimentally, it has been demonstrated that N. sativa extracts and the main constituent of their volatile oil, thymoquinone, possess antioxidant, anti-inflammatory and hepato-protective properties. To further evaluate the toxicological properties in a metabolically competent cellular system, thymoquinone was applied to primary rat hepatocyte cultures, and both cyto- and genotoxic effects were tested. Mitotic indices and the rates of apoptoses and necroses were determined as endpoints of cytotoxicity, while chromosomal aberrations and micronucleated cells served as endpoints of genotoxicity. In this approach thymoquinone demonstrated cyto- and genotoxic effects in a concentration dependent manner: it induced significant anti-proliferative effects at 20 microM and acute cytotoxicity at higher concentrations. Thymoquinone significantly increased the rates of necrotic cells at concentrations between 2.5 and 20 microM. Furthermore, it induced significant genotoxicity at concentrations > or =1.25 microM. These observations support the previous finding that thymoquinone causes glutathione depletion and liver damage, but contradict the reports indicating antioxidant and anti-clastogenic effects. Thymoquinone might be metabolised to reactive species and increase oxidative stress, which contributes to the depletion of antioxidant enzymes and damage to DNA in hepatocytes treated with high thymoquinone concentrations.
Asunto(s)
Benzoquinonas/toxicidad , Hepatocitos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Hepatocitos/citología , Mutagénesis , Necrosis/inducido químicamente , Ratas , Ratas Endogámicas F344RESUMEN
Based on a recent description of an apoptosis stimulating property for hepatocyte derived isoferritins, this investigation demonstrates that ferritin, released in vitro from hepatocytes substantially contributes to density dependent apoptosis in primary hepatocytes and is significantly (P < or = 0.05) inhibited by anti-H-ferritin antibody rH02. Furthermore, total protein release and albumin secretion rapidly decline in a time and density dependent mode under serum-free conditions, whereas ferritin secretion, which is upregulated at initial stages of primary culture is not affected by cell density. Supplementation with dexamethasone (DEX) or proliferative stimulation by epidermal growth factor (EGF) and insulin strongly suppresses density dependent apoptosis. Both regimens have previously been shown to inhibit isoferritin mediated apoptosis in hepatocytes, most likely by interrupting proapotitc mitochondrial signalling. Finally, FasL/Fas also participates in density dependent apoptosis, since apoptosis is significantly (P < or = 0.005) reduced in high density cultures supplemented with an anti-FasL antibody. This antibody has also been shown to neutralise ferritin mediated apoptosis in primary hepatocytes, suggesting a linkage of ferritin and Fas in density dependent apoptosis. In conclusion, ferritin contributes to apoptosis in primary hepatocytes in an autocrine, density dependent mode, involving Fas stimulation and proapoptotic mitochondrial signalling. With respect to liver physiology, these findings may indicate that ferritin plays a yet unrecognised role as an acute phase signalling molecule in early stages of tissue repair and liver regeneration, and may also be responsible for the limited ability to propagate human hepatocytes in culture and the limited expansion of donor cells in the recipient liver upon cell transplantation.
Asunto(s)
Apoptosis , Proteína Ligando Fas/metabolismo , Ferritinas/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Albúminas/metabolismo , Animales , Recuento de Células , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Necrosis , Pruebas de Neutralización , Ratas , Ratas Endogámicas F344 , Suero , Factores de Tiempo , Receptor fas/metabolismoRESUMEN
Aqueous extracts of Nigella sativa (Ranunculaceae) (Ns), Teucrium polium (Labiatae) (Tp) and Trigonella foenum-graecum (Fabaceae) (Tf) have been traditionally used to treat inflammations, liver disorders, and arthritis. Experimentally, it has been demonstrated that these herbs possess antioxidant, anti-inflammatory and hepatoprotective properties. To evaluate their in vitro toxicological properties and potential antimutagenic effects aqueous extracts of the three plants were tested in primary rat hepatocyte cultures against N-methyl-N'-nitro-N-nitrosoguanidine. The extracts were applied before, during and after application of MNNG to discriminate between different mechanisms of action. Tp itself significantly increased apoptosis, but in the combined treatment with MNNG significantly reduced it. Post-treatment with Ns or combined treatment with Tf significantly reduced the percentages of necrotic cells. The three plant extracts themselves significantly increased the frequency of chromosomal aberrations. Summarizing, our results suggest that aqueous extracts of the three herbs have neither cytoprotective nor antimutagenic activity, instead there is evidence for a mutagenic potential.
Asunto(s)
Alquilantes/toxicidad , Antimutagênicos/farmacología , Hepatocitos/efectos de los fármacos , Metilnitronitrosoguanidina/toxicidad , Nigella sativa , Teucrium , Trigonella , Animales , Antimutagênicos/toxicidad , Apoptosis/efectos de los fármacos , Células Cultivadas , Aberraciones Cromosómicas/inducido químicamente , Citoprotección , Relación Dosis-Respuesta a Droga , Femenino , Hepatocitos/citología , Pruebas de Mutagenicidad , Necrosis , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Plantas Medicinales , Ratas , Ratas Endogámicas F344 , Solventes , AguaRESUMEN
In order to determine whether there is a potential health risk associated with the water supply in the Aral Sea Basin, ground- and surface-water samples were collected in and around Aralsk and from the Aral Sea in 2002. Water samples from Akchi, a small town close to Almaty, served as controls. Bioassays with different toxicological endpoints were employed to assess the general toxicological status. Additionally, the samples were analysed for microbial contamination. The samples were tested in the primary hepatocyte assay for their potential to induce micronuclei and chromosomal aberrations as cumulative indicators for genotoxicity. In parallel, the effects on cell proliferation evidenced by mitotic index and cytotoxicity such as the appearance of necrotic and apoptotic cells, were determined. Furthermore, samples were examined using the Microtox assay for general toxicity. Chemical analysis according to European regulations was performed and soil and water samples were analysed for DDT and DDE. The results obtained indicated no increased cyto- or genotoxic potential of the water samples, nor levels of DDT or DDE exceeding the thresholds levels suggested by WHO. Our data therefore do not support the hypothesis that the contamination of the drinking water in and around Aralsk is responsible for the health effects previously described such as increased rates of liver disease and in particular liver cancer. Microbiological analysis, however, revealed the presence of contamination in most samples analysed.
Asunto(s)
Agua Dulce/análisis , Pruebas de Toxicidad , Abastecimiento de Agua/análisis , Animales , Células Cultivadas , Monitoreo del Ambiente/métodos , Femenino , Agua Dulce/química , Hepatocitos , Kazajstán , Luminiscencia , Metales/análisis , Pruebas de Micronúcleos , Ratas , Ratas Endogámicas F344 , Pruebas de Toxicidad/métodos , Microbiología del Agua , Contaminantes Químicos del Agua/análisisRESUMEN
Since it has to be expected that individuals exposed to oxidative stress who take supplements of beta-carotene are simultaneously exposed to both beta-carotene cleavage products (CPs) and oxidative stress, and both exposures have been demonstrated to cause genotoxic effects in primary rat hepatocytes, cyto- and genotoxic effects on primary rat hepatocytes after supplementation of the medium with increasing concentrations of a CP mixture during exposure to oxidative stress by treatment with either DMNQ (2,3-dimethoxy-1,4-naphthoquinone) or hypoxia/reoxygenation (Hy/Reox) was investigated. The cytological endpoints analysed were the mitotic indices, the percentages of apoptotic and necrotic cells, the percentages of micronucleated (MN) cells and the number of chromosomal aberrations (CAs) and sister chromatid exchanges (SCE). The results obtained clearly demonstrate that the CP mixture enhances the genotoxic effects of oxidative stress exposure, whereas it had no effect at all on the endpoints of cytotoxicity studied. These results further support the hypothesis that CP might be responsible for the reported carcinogenic response in the beta-CArotene and Retinol Efficacy Trial (CARET) and Alpha-Tocopherol Beta-carotene Cancer prevention (ATBC) chemoprevention trials.
Asunto(s)
Hepatocitos/metabolismo , beta Caroteno/fisiología , Animales , Aberraciones Cromosómicas , Daño del ADN , Relación Dosis-Respuesta a Droga , Femenino , Hipoxia , Metafase , Naftoquinonas/farmacología , Estrés Oxidativo , Oxígeno/metabolismo , Ratas , Ratas Endogámicas F344 , beta Caroteno/metabolismoRESUMEN
Free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria, a finding which could provide an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators for the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo-8'-beta-carotenal (apo-8') and beta-carotene in the primary rat hepatocyte assay in the presence and absence of oxidative stress provided by hypoxia/reoxygenation (Hy/re). The endpoints tested were: the mitotic indices, the percentages of necrotic and apoptotic cells, micronucleated cells (MN), chromosomal aberrations (CA) and sister chromatid exchanges (SCE). The results obtained indicate a genotoxic potential of both CP and apo-8' already in the concentration range of 100 nM and 1 microM, i.e. at physiologically relevant levels of beta-carotene and beta-carotene breakdown products. In contrast, no significant cytotoxic effects of these substances were observed, nor did beta-carotene induce significant cytotoxic or genotoxic effects at concentrations ranging from 0.01 up to 10 microM. However, when beta-carotene is supplemented during oxidative stress induced by hypoxia/reoxygenation, a dose-dependent increase of CP is observed accompanied by increasing genotoxicity. Furthermore, when beta-carotene cleavage products were supplied during oxidative stress significant additional increases of genotoxic effects were observed, the additional increases indicating an additive effect of both exposures. Summarizing, these results provide strong evidence that beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the beta-CArotene and RETinol Efficacy (CARET) Trial.
Asunto(s)
Mutágenos/farmacología , Estrés Oxidativo , beta Caroteno/química , beta Caroteno/farmacología , Animales , Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Aberraciones Cromosómicas , Radicales Libres/química , Hepatocitos/efectos de los fármacos , Hepatocitos/ultraestructura , Etiquetado Corte-Fin in Situ , Ratas , Intercambio de Cromátides HermanasRESUMEN
Incubation of primary cultures of parenchymal hepatocytes in a conditioned medium (CM), collected over the first 3 h of serum-free rat hepatocyte culture (CM(0-3)), induces a time dependent increase of the frequency of apoptotic cells which is accompanied by prominent changes of cell morphology. Short-term treatment with CM(0-3) for the first 3 h of culture is sufficient to significantly (P < 0.05) increase the frequency of apoptotic cells, however, the effect is more pronounced upon long-term treatment. Although apoptosis induction by CM(0-3) is independent of the timepoint when cultivation in CM(0-3) starts, our results suggest that the sensitivity for apoptosis induction by CM(0-3) is increased during the phase of attachment. Purification of CM(0-3) resulted in a fraction which significantly (P < 0.05) induced apoptosis at concentrations >/=10 ng/ml. Exposure of cultures to concentrations >/=1 microg/ml of purified CM(0-3) gave rise to a prominent cytotoxic effect as indicated by the massive occurrence of necrotic cells. Biochemical analysis showed that the purified fraction of CM(0-3) contains acidic ferritins with molecular weight of 23 and 43 kDa. Strikingly, both share homologies with placental isoferritins (PLF), for which growth inhibitory and immunosuppressive effects have been demonstrated by several investigations. Therefore, our results provide evidence that rat hepatocytes produce PLF or PLF-related acidic isoferritins which are able to induce apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Animales , Apoptosis/fisiología , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Femenino , Ferritinas/análisis , Hepatocitos/patología , Etiquetado Corte-Fin in Situ , Necrosis , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
According to Siems and colleagues, free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria. This finding may be an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators of the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo8'- carotenal (apo8') and beta-carotene utilizing primary cultures of rat hepatocytes. The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE). Our results indicate a genotoxic potential of both CP and apo8' already at the concentrations 100 nM and 1 microM, i.e. at pathophysiologically relevant levels of beta-carotene and beta-carotene breakdown products. A 3 h treatment with CP induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 10 microM and chromosomal aberrations at concentrations of 1, 5 and 10 microM. Apo8' induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 5 microM and chromosomal aberrations at concentrations of 0.1, 1 and 10 microM. Statistically significant increases in SCE induction were only observed at a concentration of 10 microM CP and apo8'. In contrast, no significant cytotoxic effects of these substances were observed. Since beta-carotene induced neither significant cytotoxic nor genotoxic effects at concentrations ranging from 0.01 up to 10 microM, these observations indicate that most likely beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET).
Asunto(s)
Antioxidantes/toxicidad , Apoptosis/efectos de los fármacos , Aberraciones Cromosómicas , Hepatocitos/efectos de los fármacos , Intercambio de Cromátides Hermanas , beta Caroteno/toxicidad , Animales , Antioxidantes/química , Femenino , Micronúcleos con Defecto Cromosómico/metabolismo , Índice Mitótico , Necrosis , Ratas , Ratas Endogámicas F344 , beta Caroteno/químicaRESUMEN
There is evidence accumulating that brain microvasculature is involved critically in oxidative stress-mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.
Asunto(s)
Apoptosis/genética , Aberraciones Cromosómicas , Endotelio Vascular/fisiopatología , Micronúcleos con Defecto Cromosómico/genética , Estrés Oxidativo/genética , Animales , Western Blotting , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Glucosa/metabolismo , Hipoxia/genética , L-Lactato Deshidrogenasa/análisis , Naftoquinonas/farmacología , Ratas , Daño por Reperfusión/genética , Porcinos , Telencéfalo/irrigación sanguínea , Telencéfalo/efectos de los fármacos , Telencéfalo/fisiopatología , Factores de Tiempo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
PURPOSE: To advance a biomathematical model of radiocarcinogenesis by describing multiple pathways for initiation, a radiologically induced cell-cycle delay, misrepair and spontaneous DNA damages caused by replication. It was investigated whether the incorporation of these biological features would improve the fit of the model to data showing plateaus in in vitro irradiations of different cell lines and whether the fit parameters were then more biologically realistic. MATERIALS AND METHODS: A biomathematical submodel was developed based on a previous State-Vector Model that mathematically described enhanced DNA repair and radical scavenging following irradiation. RESULTS: With the two initiation pathways and cell-cycle delay, the simulations better explained the mouse data but not the rat data, and for both data sets the fit parameters were biologically more realistic than previously assumed. Inclusion of misrepair and replicational errors did not significantly affect the fit. CONCLUSIONS: A plateau in the dose-effect relationship for in vitro irradiation of different cell lines can be explained by radioprotective mechanisms. The plateau-type dose-response relationships point to a non-linear dose- effect relationship at low doses and indicate that linear extrapolation from moderate (or high) to low doses may not be justified for in vitro studies of these cell lines.
Asunto(s)
Ciclo Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Animales , Aberraciones Cromosómicas , Relación Dosis-Respuesta en la Radiación , Femenino , Transferencia Lineal de Energía , Ratones , Ratones Endogámicos C3H , Ratas , Ratas Endogámicas F344RESUMEN
Blood samples were irradiated in vitro with alpha-rays emitted from short-lived radon decay products dissolved in the culture medium at doses between 0.03 and 41.4 mGy. The data were collected from experiments conducted during the period 1984-1992 and comprise a total of about 64000 scored metaphases. For statistical reasons, only 60,022 metaphases were used for the subsequent analysis. The results for total chromosome aberrations and dicentrics indicate a linear dose dependence in the dose range above about 10 mGy, consistent with other experimental observations. At doses below about 10 mGy, aberration frequencies cannot be linearly extrapolated from higher doses, suggesting that there is no dependence on dose within a certain low-dose range. In addition, a statistically significant minimum has been observed at a dose of about 0.03 mGy, which is consistently lower than the related control values. The behavior of the aberration frequencies in the low-dose region seems to be influenced by the control values, which also depend on the environmental radiation burdens to the donors before blood sampling and thus were significantly affected by the Chernobyl fallout.
Asunto(s)
Partículas alfa , Aberraciones Cromosómicas , Linfocitos/efectos de la radiación , Radón/toxicidad , Relación Dosis-Respuesta en la Radiación , Humanos , Peroxidación de Lípido/efectos de la radiación , Linfocitos/ultraestructura , Centrales Eléctricas , Liberación de Radiactividad Peligrosa , Factores de Tiempo , UcraniaRESUMEN
PURPOSE: To investigate whether it is possible to explain dose-response plateaus for in-vitro X-ray irradiation of different cell lines with radioprotective mechanisms such as radiologically induced expression of scavengers and repair enzymes. MATERIALS AND METHODS: A biomathematical model was developed based on a previous state-vector model. New features of the model are a mathematical description of enhanced repair and radical scavenging as a result of irradiation. RESULTS: The model produces a plateau in the dose-response for in-vitro tranformations between 0.5 and 1 Gy and for chromosome aberrations and it predicts an inverse-fractionation effect within a selected range of doses. CONCLUSIONS: Adaptive response mechanisms within a state-vector model provide a coherent explanation of the dose-response characteristics for in-vitro transformations and chromosomal aberrations. These results suggest the need for new experimental studies described in the paper.
Asunto(s)
Adaptación Biológica/fisiología , Modelos Biológicos , Neoplasias Inducidas por Radiación/etiología , Tolerancia a Radiación/fisiología , Animales , Transformación Celular Neoplásica/efectos de la radiación , ADN/efectos de la radiación , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Cómputos Matemáticos , Ratones , Modelos Estadísticos , Neoplasias Inducidas por Radiación/genética , Rayos XRESUMEN
INTRODUCTION: This report describes the first study on genotoxic effects of Austrian ground- and drinking waters. Samples from the Mitterndorfer Senke (MS) and the vicinity of Wiener Neustadt were tested over a three years period. The MS is the largest aquifer in Austria. Due to deposition of industrial and community wastes, chemicals have polluted the groundwater in this area. Aim of the present study was to elucidate if consumption of these waters might pose a carcinogenic risk to humans. METHODS: 43 Water samples were tested in a test battery which consisted of bacterial gene mutation assays (Salmonella strains TA100 and TA98), micronucleus (MN) assays with cultures of primary rat hepatocytes and plant bioassays (MN tests with Tradescantia and Vicia faba). For the bacterial assays, the water samples were extracted with XAD resins. RESULTS: In total, 27.9% of the samples caused positive effects; 8 samples were active in Salmonella microsome assays, Strain TA100 was particularly sensitive upon addition of metabolic activation mix (6 positive samples). Four samples were positive exclusively in MN assays with cultures of primary rat hepatocytes; one sample gave positive results in all three bioassays. Finished waters from waterworks were consistently devoid of mutagenic activity under all experimental conditions. DISCUSSION: Overall, only a small fraction of the groundwaters caused mutagenic effects and in all cases the activities were moderate. Comparison of the results of the present study with data obtained in other investigations under similar experimental conditions shows that the genotoxicity of groundwaters of the MS area are lower than the effects caused by ground- and drinking waters from other countries. The fact that no genotoxic activity was detected in any of the finished drinking waters can be taken as an indication that consumption of these waters does not pose a health hazard arising from contamination with genotoxic carcinogens to humans.
Asunto(s)
Pruebas de Mutagenicidad , Mutágenos/efectos adversos , Contaminantes Químicos del Agua/efectos adversos , Animales , Humanos , Concentración Máxima Admisible , Mutágenos/análisis , Ratas , Factores de Riesgo , Contaminantes Químicos del Agua/análisis , Abastecimiento de Agua/análisisRESUMEN
Recent advances in cell culture have allowed proliferation of primary rat hepatocytes enabling the analysis of different cytogenetic endpoints such as sister chromatid exchanges (SCE), chromosomal aberrations and micronuclei. The latter are of particular interest as preparation, staining and analysis is less time-consuming than the analysis of chromosomal aberrations and SCE what makes micronuclei an attractive short-term assay. This paper gives (1) a summary of the specific features of primary hepatocytes including ploidy, nuclearity and multipolar mitoses, (2) a summary of the culture conditions for proliferation and the proliferation kinetics, (3) an experimentally based interpretation of the comparatively high background levels of cells with micronuclei, and (4) an experiment-based discussion of approaches to circumvent the major disadvantage: the cytochalasin B method cannot be applied due to the high percentage of binucleated cells.