RESUMEN
Sterigmatocystin (STE) is a common mycotoxin found in food and feed. Many studies showed that STE is genotoxic. However, up to now, the potential genotoxicity of STE on human neuronal system remains unknown. In this study, we explored the effect of STE on DNA damage and cell-cycle progression on human neuroblastoma SH-SY5Y cells exposed to various concentrations of STE (0.78, 1.56 and 3.12 µM) for 24 h. The results indicated that STE exposure induced DNA damage, as evidenced by DNA comet tails formation and increased γH2AX foci. Additionally, genotoxicity was confirmed by micronuclei (MN) analysis. Furthermore, we found that STE exposure led to cell-cycle arrest at the S and the G2/M phase. Considering the important role played by MAPK and p53 signaling pathways in cell-cycle arrest, we explored their potential involvement in STE-induced cell-cycle arrest by using specific inhibitors. The inhibition of JNK and ERK resulted to attenuate S and G2/M arrest, whereas the inhibition of p38 and p53 attenuated only STE-induced S phase arrest. In conclusion, the present study demonstrates that STE induced DNA damage and triggered MAPK and p53 pathways activation, resulting in cell-cycle arrest at the S and the G2/M phase.
Asunto(s)
Neuroblastoma , Apoptosis , Puntos de Control del Ciclo Celular , Daño del ADN , Humanos , Transducción de Señal , Esterigmatocistina/toxicidad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis following inactivation of Chk1. Taken together, we show that ST treatment for 24 h activates Chk1 and induces a G2 arrest in GES-1 cells. However, prolonged ST treatment for 48 h led to Chk1 inactivation in GES-1 cells, which promotes checkpoint adaptation and entry of cells into mitosis despite damaged DNA. Importantly, checkpoint adaptation in GES-1 cells treated with ST may potentially promote genomic instability and drive tumorigenesis.
Asunto(s)
Carcinogénesis/inducido químicamente , Carcinógenos Ambientales/toxicidad , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Mucosa Gástrica/efectos de los fármacos , Mitosis/efectos de los fármacos , Esterigmatocistina/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Activación Enzimática/efectos de los fármacos , Fase G2/efectos de los fármacos , Mucosa Gástrica/citología , Mucosa Gástrica/patología , Inestabilidad Genómica/efectos de los fármacos , Humanos , Cinética , Índice Mitótico , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various contaminants or groups of contaminants in food. The first part of the report contains a brief description of general considerations addressed at the meeting, including updates on matters of interest to the work of the Committee. A summary follows of the Committee's evaluations of technical, toxicological and/or dietary exposure data for six contaminants or groups of contaminants (aflatoxins, 4,15-diacetoxyscirpenol, fumonisins, glycidyl esters, 3-MCPD esters and 3-MCPD, sterigmatocystin) as well as an evaluation of co-exposure of fumonisins with aflatoxins. Annexed to the report is a summary of the toxicological and dietary exposure information as well as the Committee's recommendations on the contaminants and groups of contaminants considered at this meeting.
Asunto(s)
Contaminación de Alimentos/análisis , Microbiología de Alimentos , Inocuidad de los Alimentos , Aflatoxinas/toxicidad , Compuestos Epoxi/toxicidad , Ésteres , Fumonisinas/toxicidad , Humanos , Esterigmatocistina/toxicidad , Tricotecenos/toxicidad , Organización Mundial de la Salud , alfa-Clorhidrina/toxicidadRESUMEN
Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.
Asunto(s)
Daño del ADN/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Esófago/efectos de los fármacos , Esterigmatocistina/toxicidad , Antígenos Transformadores de Poliomavirus/genética , Línea Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/metabolismo , Esófago/citología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
1. The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin. 2. The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity. 3. CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity. 4. These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Células Epiteliales/metabolismo , Redes y Vías Metabólicas/fisiología , Esterigmatocistina/análogos & derivados , Tráquea/citología , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Humanos , Estructura Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Esterigmatocistina/química , Esterigmatocistina/metabolismo , Esterigmatocistina/toxicidad , Porcinos , Espectrometría de Masas en TándemRESUMEN
This study investigates the individual and combined effects of the mycotoxins, Aflatoxin B1 (AFB1), Enniatin B (ENNB) and Sterigmatocystin (STG), on the cellular viability of gastric (NCI-N87), intestinal (Caco-2), hepatic (Hep-G2) and renal (Hek-293) cells, shedding light on synergistic or antagonistic effects using a constant ratio combination design proposed by Chou-Talalay. These toxins are prevalent in cereal-based foods, frequently consumed by children which raises concerns about their exposure to these mycotoxins. This population is particularly vulnerable to the effects of these toxins due to their underdeveloped organs and incompletely structured physiological processes. Results showed that ENB was the most toxic of the three mycotoxins across all cell lines, while STG and AFB1 showed lower toxicity. The combination of ENNB + STG was found to be the most potent in terms of binary mixtures. In regard to ternary combinations, Caco-2 cells are more sensitive to the tested mycotoxins, whereas NCI-N87 cells show lower levels of cell damage. Worrying dose reduction values (>10-fold) were found for ENNB in binary and ternary combinations at low exposure levels. These findings are significant for establishing initial reference values, which play a pivotal role in estimating reference doses that are subsequently incorporated into the broader risk assessment process.
Asunto(s)
Aflatoxina B1 , Depsipéptidos , Esterigmatocistina , Humanos , Esterigmatocistina/toxicidad , Aflatoxina B1/toxicidad , Depsipéptidos/toxicidad , Supervivencia Celular/efectos de los fármacos , Células CACO-2 , Hígado/efectos de los fármacos , Riñón/efectos de los fármacos , Intestinos/efectos de los fármacos , Células HEK293 , Células Hep G2RESUMEN
Mycotoxins are natural food and feed contaminants produced by several molds. The primary mode of exposure in humans and animals is through mixtures. Aflatoxin B1 (AFB1) and sterigmatocystin (STER) are structurally related mycotoxins that share the same biosynthetic route. Few in vivo genotoxicity assays have been performed with STER. In the present genotoxicity study, Wistar rats were dosed orally with STER (20 mg/kg b.w.), AFB1 (0.25 mg/kg b.w.) or a mixture of both in an integrated micronucleus (bone marrow) and comet study (liver and kidney). STER was dosed at the highest feasible dose in corn oil. No increase in the percentage of micronuclei in bone marrow was observed at any condition. Slight DNA damage was detected in the livers of animals treated with AFB1 or the mixture (DNA strand breaks and Fpg (Formamidopyrimidine DNA glycosylase)-sensitive sites, respectively). Plasma, liver, and kidney samples were analyzed with LC-MS/MS demonstrating exposure to both mycotoxins. General toxicity parameters (organs absolute weight, biochemistry, and histopathology) were not altered either individually or in the mixture. The overall absence of individual genotoxicity did not allow us to set any type of interaction in the mixture. However, a possible toxicokinetic interaction was observed.
Asunto(s)
Aflatoxina B1 , Esterigmatocistina , Ratas , Animales , Humanos , Ratas Wistar , Esterigmatocistina/toxicidad , Aflatoxina B1/toxicidad , Cromatografía Liquida , Espectrometría de Masas en Tándem , Daño del ADNRESUMEN
Aspergillus versicolor and A. flavus are primary colonizers in damp dwellings, and they produce sterigmatocystin (ST) and aflatoxin B1 (AFB(1)), respectively. These hepatotoxic and carcinogenic mycotoxins and their precursors and derivates possess a furofuran ring, which has proven responsible for their toxicity. The aim of this study was to investigate the cytotoxicity and genotoxicity of versicolorin A (VER A) and versicolorin B (VER B), as the furofuran precursors of aflatoxins and ST, and of 5-methoxysterigmatocystin (5-MET-ST), a methoxy derivative of ST, in human adenocarcinoma lung cells A549. The IC(50) values of the tested compounds were obtained by the cell proliferation MTT test as follows: 109 ± 3.5 µM (VER A), 172 ± 4 µM (VER B) and 181 ± 2.6 µM (5-MET-ST). The comet assay and micronucleus test were used to assess their genotoxic potential after 24 h of treatment with concentrations corresponding to ½ and » IC(50) in comparison with AFB(1) and ST, applied in concentrations corresponding to ½ IC(50), as previously determined in A549 cells. DNA damage parameters assessed by the comet assay were tail length, tail intensity and tail moment, while the level of DNA damage in the micronucleus test was evaluated by the number of formed micronuclei (MN), nuclear buds (NB) and nucleoplasmic bridges (NPB) in 1,000 binucleated cells. Considering the three comet parameters, all applied toxins exerted significant DNA damage compared to the control, while ST and VER B produced the highest DNA damage. All toxins provoked a statistically significant increase in MN, and a slightly decreased formation of NB and NPB. AFB(1), ST and 20 µM VER A showed a statistically significant increase in all three micronucleus parameters compared to the control, and the highest increase in the number of MN occurred in cells treated with 50 µM VER A. The differences between results obtained by the micronucleus test and comet assay could be explained by the fact that the micronucleus detects irreversible DNA damage, which is usually correlated with the previously determined cytotoxic potential of the AFB(1) precursors.
Asunto(s)
Antraquinonas/toxicidad , Mutágenos/toxicidad , Esterigmatocistina/análogos & derivados , Adenocarcinoma/metabolismo , Antraquinonas/administración & dosificación , Aspergillus/química , Aspergillus flavus/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/metabolismo , Pruebas de Micronúcleos , Mutágenos/administración & dosificación , Esterigmatocistina/administración & dosificación , Esterigmatocistina/toxicidad , Factores de TiempoRESUMEN
Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.
Asunto(s)
Aflatoxinas , Esterigmatocistina , Aflatoxina B1/toxicidad , Animales , Escherichia coli , Microinyecciones , Esterigmatocistina/toxicidad , Pez CebraRESUMEN
The toxicity of mycotoxins containing bisfuranoid structures such as aflatoxin B1 (AFB1) depends largely on biotransformation processes. While the genotoxicity and mutagenicity of several bisfuranoid mycotoxins including AFB1 and sterigmatocystin have been linked to in vivo bioactivation of these molecules into reactive epoxide forms, the metabolites of genotoxic and mutagenic AFB1 precursor versicolorin A (VerA) have not yet been characterized. Because this molecule is not available commercially, our strategy was to produce a library of metabolites derived from the biotransformation of in-house purified VerA, following incubation with human liver S9 fractions, in presence of appropriate cofactors. The resulting chromatographic and mass-spectrometric data were used to identify VerA metabolites produced by intestinal cell lines as well as intestinal and liver tissues exposed ex vivo. In this way, we obtained a panel of metabolites suggesting the involvement of phase I (M + O) and phase II (glucuronide and sulfate metabolites) enzymes, the latter of which is implicated in the detoxification process. This first qualitative description of the metabolization products of VerA suggests bioactivation of the molecule into an epoxide form and provides qualitative analytic data to further conduct a precise metabolism study of VerA required for the risk assessment of this emerging mycotoxin.
Asunto(s)
Aflatoxina B1 , Aflatoxinas , Aflatoxina B1/metabolismo , Aflatoxina B1/toxicidad , Aflatoxinas/toxicidad , Antraquinonas , Daño del ADN , Compuestos Epoxi , Humanos , Mutágenos/toxicidad , Esterigmatocistina/toxicidadRESUMEN
OBJECTIVE: The causal agents of gastric cancer could include fungus toxins. Sterigmatocystin (ST), a fungus toxin, is a risk factor of gastric cancer. We investigated the effects of ST on the stomach tissues of Mongolian gerbils. METHODS: Seventy-five-week-old male Mongolian gerbils received ST ad libitum at a concentration of 0 ppb (non-treated, n = 11), 100 ppb (n = 7), or 1000 ppb (n = 13) dissolved in drinking water for a period of 24 weeks. After administration, we tested the histopathological changes and immunostaining for proliferating cell nuclear antigen (PCNA), p53, and MDM2 expression. RESULTS: We investigated the histopathological changes and determined the incidence of histopathological changes in animals with various gastric diseases after ST administration at a dose of 0 ppb (non-treated control), 100, or 1,000 ppb as follows: firstly, indices for gastritis were 18.2, 100, and 100%, those for erosion events were 9.1, 100, and 92.3%, and those for polyps were 0, 71.4, and 61.5%, respectively. These incidences in the ST-administered groups (100 or 1000 ppb) showed significant increases compared with those in the non-treated control group. And, lastly, indices for intestinal metaplasia were 0, 100, and 15.4%, respectively. Furthermore, immunostaining for PCNA, p53, and MDM2 expression showed significantly greater rates in the ST-administered groups (100 or 1000 ppb) than in the non-treated control group. CONCLUSION: The histopathological and immunohistopathological findings of this study indicate that ST exerts a marked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.
Asunto(s)
Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/patología , Gastritis/inducido químicamente , Esterigmatocistina/toxicidad , Animales , Aspergillus/efectos de los fármacos , Modelos Animales de Enfermedad , Mucosa Gástrica/microbiología , Gastritis/microbiología , Gastritis/patología , Gerbillinae , Masculino , Metaplasia/inducido químicamente , Metaplasia/microbiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Organismos Libres de Patógenos Específicos , Esterigmatocistina/administración & dosificación , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.
Asunto(s)
Quinasa de Punto de Control 2/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Mutágenos/toxicidad , Esterigmatocistina/análogos & derivados , Células A549 , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Células Hep G2 , Humanos , Fosforilación/efectos de los fármacos , Esterigmatocistina/toxicidadRESUMEN
Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour.
Asunto(s)
Técnicas de Cultivo Tridimensional de Células/métodos , Micotoxinas/toxicidad , Esterigmatocistina/toxicidad , Pruebas de Toxicidad/métodos , Western Blotting , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ensayo Cometa/métodos , Técnica del Anticuerpo Fluorescente , Humanos , Neuroblastoma , Especies Reactivas de Oxígeno/metabolismo , Esferoides Celulares/efectos de los fármacosRESUMEN
Oxidative stress appears to be a common trigger for many of the effects associated with the exposure to various mycotoxins, including sterigmatocystin (STE). However, studies to alleviate STE toxicity through the use of natural antioxidants are sparsely reported in literature. In the present study, the cytoprotective effect of quercetin (QUE) was tested in SH-SY5Y cells against STE-induced oxidative stress and cytotoxicity. The MTT assay revealed that STE decreased cell viability, whereas pre-treatment of cells with QUE restored it. The QUE was also found to counteract STE-induced ROS generation and decrease STE-induced up-regulation of the expression of the stress-inducible enzymes HO-1 and NOS-2. Pre-treatment with QUE also prevented STE-induced nuclear translocation of NF-κB, as measured by immunofluorescence. Finally, considering the key role played by NF-κB in the regulation of inflammation, the effect of STE on the pro-inflammatory cytokines TNF-α and IL-6 expression was evaluated. Our results showed the down-regulation of TNF-α and IL-6 following STE exposure, suggesting a negative immunomodulatory effect of STE. In QUE pre-treated samples, TNF-α and IL-6 were significantly further reduced, indicating the anti-inflammatory role of QUE. The results of the present study demonstrate for the first time that QUE exerts a cytoprotective role in STE-induced toxicity.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Quercetina/farmacología , Esterigmatocistina/toxicidad , Antioxidantes/farmacología , Biomarcadores/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunomodulación/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismoRESUMEN
The purpose of the study was to evaluate the short-term effects of aflatoxin B1 (AFB1 100 µg/kg feed) and sterigmatocystin (STC 1000 µg/kg feed) exposure individually and in combination (100 µg AFB1 + 1000 µg STC/kg feed) on the parameters of lipid peroxidation and glutathione redox system both in biochemical and gene expression levels in one-year-old common carp. Lipid peroxidation parameters were slightly affected, as significant differences were observed only in conjugated diene and triene concentrations. Reduced glutathione content decreased more markedly by STC than AFB1 or AFB1+STC, but glutathione peroxidase activity did not change. Expression of gpx4a, gpx4b, gss, and gsr genes was down-regulated due to STC compared to AFB1 or AFB1+STC, while an induction was found as effect of AFB1+STC in the case of gpx4a, but down-regulation for gpx4b as compared to AFB1. Expression of the glutathione biosynthesis regulatory gene, gss, was higher, but glutathione recycling enzyme encoding gene, gsr, was lower as an effect of AFB1+STC compared to AFB1. These results are supported by the changes in the expression of transcription factors encoding genes, nrf2, and keap1. The results revealed that individual effects of AFB1 and STC on different parameters are synergistic or antagonistic in multi-toxin treatment.
Asunto(s)
Aflatoxina B1/toxicidad , Carpas/metabolismo , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Esterigmatocistina/toxicidad , Animales , Carpas/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Glutatión Sintasa/genética , Glutatión Sintasa/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismoRESUMEN
Exposure to sterigmatocystin (STC) raises concerns on developmental neurological disorders. The present study investigated the effects of maternal oral STC exposure on postnatal hippocampal neurogenesis of offspring in rats. Dams were exposed to STC (1.7, 5.0, and 15.0 ppm in diet) from gestational day 6 until day 21 post-delivery (weaning), and offspring were maintained without STC exposure until adulthood on postnatal day (PND) 77, in accordance with OECD chemical testing guideline Test No. 426. On PND 21, 15.0-ppm STC decreased type-3 neural progenitor cell numbers in the subgranular zone (SGZ) due to suppressed proliferation. Increased γ-H2AX-immunoreactive (+) cell numbers in the SGZ and Ercc1 upregulation and Brip1 downregulation in the dentate gyrus suggested induction of DNA double-strand breaks in SGZ cells. Upregulation of Apex1 and Ogg1 and downregulation of antioxidant genes downstream of NRF2-Keap1 signaling suggested induction of oxidative DNA damage. Increased p21WAF1/CIP1+ SGZ cell numbers and suppressed cholinergic signaling through CHRNB2-containing receptors in GABAergic interneurons suggested potential neurogenesis suppression mechanisms. Multiple mechanisms involving N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling and various GABAergic interneuron subpopulations, including CHRNA7-expressing somatostatin+ interneurons activated by BDNF-TrkB signaling, may be involved in ameliorating the neurogenesis. Upregulation of Arc, Ptgs2, and genes encoding NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors suggested synaptic plasticity facilitation. On PND 77, ARC+ granule cells decreased, and Nos2 was upregulated following 15.0 ppm STC exposure, suggesting oxidative stress-mediated synaptic plasticity suppression. Inverse pattern in gene expression changes in vesicular glutamate transporter isoforms, Slc17a7 and Slc17a6, from weaning might also be responsible for the synaptic plasticity suppression. The no-observed-adverse-effect level of maternal oral STC exposure for offspring neurogenesis was determined to be 5.0 ppm, translating to 0.34-0.85 mg/kg body weight/day.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Esterigmatocistina/toxicidad , Animales , Apoptosis/efectos de los fármacos , Roturas del ADN de Doble Cadena , Giro Dentado/metabolismo , Giro Dentado/patología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Nivel sin Efectos Adversos Observados , Estrés Oxidativo/efectos de los fármacos , Ratas Sprague-Dawley , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/metabolismo , DesteteRESUMEN
Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with (13)C to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by real-time quantitative real-time polymerase chain reaction. Incubation of 50 µM sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 µM sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with ß-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxy-sterigmatocystin. Exposure of PTEC for 24 h to 1 µM sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in the porcine pulmonary tract need to be confirmed in human epithelial cells.
Asunto(s)
Células Epiteliales/metabolismo , Esterigmatocistina/metabolismo , Tráquea/citología , Animales , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Humanos , Inactivación Metabólica , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esterigmatocistina/farmacocinética , Esterigmatocistina/toxicidad , PorcinosRESUMEN
Mitochondria are cellular organelles involved in many crucial functions, such as generation of energy (ATP) and initiation of apoptosis. The aim of the present study was to evaluate the role of mitochondria in the toxicity induced by sterigmatocystin (STE), a mycotoxin produced by fungi of the genus Aspergillus, on SH-SY5Y cells. Our results showed that STE exposure decreased cell viability in a time- and concentration-dependent manner by MTT assay and caused mitochondrial dysfunction, as highlighted by the increase of STE cytotoxicity in cells forced to rely on mitochondrial oxidative phosphorylation. Furthermore, intracellular ATP depletion and increased mitochondrial reactive oxygen species were also observed. Since mitochondria play a pivotal role in apoptosis, the induction of this process in response to STE exposure was decided to study. Our results showed an increase in apoptotic cell population by flow cytometry, further confirmed by the up-regulation of the expression levels of the pro-apoptotic genes Bax and Casp-3 and the down-regulation of the anti-apoptotic gene Bcl-2 by qPCR technique. Taken together, our results provide novel insights in the signalling pathways of the cell death process induced by STE in SH-SY5Y cells, highlighting the key role played by mitochondria in STE toxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Esterigmatocistina/toxicidad , Adenosina Trifosfato/metabolismo , Caspasa 3/genética , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Esterigmatocistina/administración & dosificación , Proteína X Asociada a bcl-2/genéticaRESUMEN
Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are mycotoxins produced by common damp indoor Aspergilli series Versicolores. Since both STC and 5-M-STC were found in the dust of indoor occupational and living areas, their occupants may be exposed to these mycotoxins, primarily by inhalation. Thus, STC and 5-M-STC were intratracheally instilled in male Wistar rats using doses (0.3 mg STC/kg of lung weight (l.w.); 3.6 mg 5-M-STC/kg l.w.; toxin combination 0.3 + 3.6 mg/kg l.w.) that corresponded to concentrations detected in the dust of damp indoor areas in order to explore cytotoxicity, vascular permeability, immunomodulation and genotoxicity. Single mycotoxins and their combinations insignificantly altered lactate-dehydrogenase activity, albumin, interleukin-6, tumor necrosis factor-α and chemokine macrophage inflammatory protein-1α concentrations, as measured by ELISA in bronchioalveolar lavage fluid upon 24 h of treatment. In an alkaline comet assay, both mycotoxins provoked a similar intensity of DNA damage in rat lungs, while in a neutral comet assay, only 5-M-STC evoked significant DNA damage. Hence, naturally occurring concentrations of individual STC may induce DNA damage in rat lungs, in which single DNA strand breaks prevail, while 5-M-STC was more responsible for double-strand breaks. In both versions of the comet assay treatment with STC + 5-M-STC, less DNA damage intensity occurred compared to single mycotoxin treatment, suggesting an antagonistic genotoxic action.
Asunto(s)
Pulmón/efectos de los fármacos , Mutágenos/toxicidad , Esterigmatocistina/análogos & derivados , Albúminas/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Ensayo Cometa , Citocinas/metabolismo , Daño del ADN , Interacciones Farmacológicas , L-Lactato Deshidrogenasa/metabolismo , Pulmón/metabolismo , Masculino , Ratas Wistar , Esterigmatocistina/toxicidadRESUMEN
Sterigmatocystin (STE) is a mycotoxin produced by fungi of the genus Aspergillus. Considering that the effect of STE on neuronal system has not been well studied, the aim of the present study consists to investigate the cytotoxic effects of STE in human neuroblastoma (SH-SY5Y) cells. Moreover, the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and antioxidant no-enzymatic (GSH) levels and enzymatic (GPx, GST, CAT and SOD) activity. Our results revealed that STE decreased cell viability in a dose and time-dependent manner. Furthermore, after 24â¯h of exposure, STE induced an increase in ROS generation and LPO at all concentrations tested (0.78, 1.56 and 3.12⯵M), as well as a depletion of GSH levels, an increase in GSSG content and a decrease in GSH/GSSG ratio at the highest concentrations. The activity of all antioxidant enzymes resulted to be also decreased. Additionally, an enhance of the oxidative damage was caused by BSO, a GSH depletor, while NAC, a GSH precursor, showed a scavenger activity. Our findings suggest that STE could injure SH-SY5Y cells via oxidative stress and highlight the antioxidant role of the glutathione system.