Investigating the individual and mixture cytotoxicity of co-occurring aflatoxin B1, enniatin B, and sterigmatocystin on gastric, intestinal, hepatic, and renal cellular models.

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.

In Vivo Genotoxicity and Toxicity Assessment of Sterigmatocystin Individually and in Mixture with Aflatoxin B1.

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.

Metabolism of versicolorin A, a genotoxic precursor of aflatoxin B1: Characterization of metabolites using in vitro production of standards.

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.

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

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.

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing.

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.

Role of quercetin on sterigmatocystin-induced oxidative stress-mediated toxicity.

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.

Sterigmatocystin, 5-Methoxysterigmatocistin, and Their Combinations Are Cytotoxic and Genotoxic to A549 and Hepg2 Cells and Provoke Phosphorylation of Chk2, but Not Fancd2 Checkpoint Proteins.

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.

Disruption of postnatal neurogenesis and adult-stage suppression of synaptic plasticity in the hippocampal dentate gyrus after developmental exposure to sterigmatocystin in rats.

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.

Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells.

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.

Individual and Combined Effects of Aflatoxin B1 and Sterigmatocystin on Lipid Peroxidation and Glutathione Redox System of Common Carp Liver.

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.

Single-Dose Toxicity of Individual and Combined Sterigmatocystin and 5-Methoxysterigmatocistin in Rat Lungs.

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.

Sterigmatocystin: Occurrence, toxicity and molecular mechanisms of action - A review.

The mycotoxin sterigmatocystin (STE) is produced mainly by Aspergillus fungi. It has been reported to occur in grains and grain-based products, cheese, coffee, spices and beer. The STE is a known biogenic precursor of aflatoxin B1, sharing with it several structural and biological similarities. The STE has been shown to be hepatotoxic and nephrotoxic in animals and it has been classified as possible human carcinogen (group 2B) by IARC. The STE has been reported to cause a marked decrease in cell proliferation in different mammalian cells. Data available on literature suggest that the cellular mechanisms underlying STE-induced toxicity include the induction of oxidative stress, mitochondrial dysfunction, apoptosis, cell cycle arrest, as well as alteration of immune system function and activation of different signalling pathways. Moreover, STE resulted to be genotoxic, being able to form DNA-adducts and induce DNA damage. Despite its strong cytotoxicity, no risk assessments have been still carried out by authorities due to the lack of toxicity data, so research on STE toxicological impact is still going on. This review reports information available regarding STE toxicity and its related mechanisms of action with the aim of updating information regarding last researches on this mycotoxin.

The role of mitochondria in sterigmatocystin-induced apoptosis on SH-SY5Y cells.

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.

Sterigmatocystin moderately induces oxidative stress in male Wistar rats after short-term oral treatment.

This study aimed to explore involvement of oxidative stress in sterigmatocystin (STC) toxicity in male Wistar rats. Animals were orally treated with a single STC dose (10, 20 and 40 mg/kg b.w.). Short-term treatment resulted in moderate oxidative stress determined by a significant increase of malondialdehyde (MDA; all STC doses) and catalase (CAT; 10 mg/kg b.w.) in plasma, a decrease of glutathione peroxidase (GPx; 20 and 40 mg/kg b.w.) in the liver, and increase of MDA and superoxide dismutase (SOD) in kidneys (all STC doses). Heat shock protein (Hsp27 and Hsp70) expression was determined by Western blotting in rat liver and kidneys. Hsp27 expression was downregulated by STC, particularly in the liver (40 mg/kg b.w.). The lowest STC dose elevated the expression of Hsp70 in both liver and kidneys, while an increase in STC doses restored Hsp70 expression to control. Alterations in expressions of Hsp27 and Hsp70 could be only partially associated with oxidative stress. STC provoked a significant DNA damage in both liver and kidneys (alkaline comet assay), but the liver was more affected by a broader spectrum of DNA lesions. Oxidative DNA damage (hOGG1-modified comet assay) contribute to the overall mechanism of STC-induced DNA damage in both organs, but kidneys in general seem to be more susceptible to oxidative stress upon short-term exposure to sublethal doses of STC.

Sterigmatocystin-induced cytotoxicity via oxidative stress induction in human neuroblastoma cells.

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 24 h of exposure, STE induced an increase in ROS generation and LPO at all concentrations tested (0.78, 1.56 and 3.12 µM), 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.

Sterigmatocystin: A mycotoxin to be seriously considered.

Sterigmatocystin is a carcinogenic compound that affects several species of crops and several species of experimental animals. The sterigmatocystin biosynthetic pathway is the best known and most studied. The International Agency for Research on Cancer classifies sterigmatocystin in the Group 2B. Three groups of analytical methods to determine sterigmatocystin in food can be found: chromatographic, ELISA immunoassays and chemical sensors. In addition, sterigmatocystin is a precursor of aflatoxin B1 in those cases where cereals and/or food are contaminated with fungi capable of producing aflatoxins. Chemical structures of sterigmatocystin and aflatoxin B1 are similar. These mycotoxins are pathogens of animals and cereals, producing a major economic impact on biotechnology and agricultural and food industries. This review summarizes different aspects related to sterigmatocystin such as its biosynthesis, toxicological studies and analytical methods for its determination.

Genotoxicity of aflatoxins and their precursors in human cells.

Aflatoxins are found as food contaminant and some of them demonstrate a carcinogenic effect. The aflatoxins biosynthetic pathway involves 15 successive steps. The aim of this study was to compare the toxicity of aflatoxins and their precursors in three human cell lines. We tested the four aflatoxins and two of their metabolites; three early metabolic precursors and two late biosynthetic precursors. Cyclopiazonic acid, synthesized in parallel with aflatoxins, was also tested. The cytotoxicity and the genotoxicity was evaluated with the γH2AX assay in three human cell lines with different bioactivation capacities. Our results indicated that the most genotoxic chemicals in the three cell lines were in decreasing order sterigmatocystin (ST), aflatoxin B1 (AFB1), aflatoxicol (AFL), aflatoxin G1 (AFG1) and versicolorin A (VERA). Aflatoxin M1 (AFM1) demonstrated genotoxic property in only one cell line. The other tested compounds did not demonstrate any genotoxic activity. Overall, our results suggested different genotoxic mechanisms of action for the tested compounds, involving specific bioactivation pathways. Moreover, some metabolic precursors of aflatoxins demonstrated genotoxic potential equivalent or greater to AFB1. This should be taking into account for the development of new strategies intended to reduce the aflatoxins exposure and for human risk assessment.

Evaluation of certain contaminants in food.

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.

Sterigmatocystin induced apoptosis in human pulmonary cells in vitro.

Sterigmatocystin (ST) is generally recognized as a potential carcinogen, mutagen and teratogen. Studies showed that ST could induce adenocarcinoma of lung in mice in vivo and DNA damage, cell cycle arrest in a human immortalized bronchial epithelial cell line (BEAS-2B cells) and a human lung cancer cell line (A549 cells) in vitro. Besides, ST could induce G2 arrest (cell cycle arrest in G2 phase) in several other cells. Cell cycle arrest may be one of the common toxic effects of ST. As cells may undergo apoptosis or death due to cell cycle arrest, we wondered whether apoptosis is another common effect of ST in different cells in vitro. In the present study, we studied the effects of ST on proliferation and apoptosis in A549 cells and BEAS-2B cells with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis (FCM). The MTT results showed that proliferation inhibition following ST treatment for 24h was observed in both A549 and BEAS-2B cells in vitro. And increased apoptosis by FCM was also found after ST treatment. Down-regulation of Bcl-2, up-regulation of Bax and the activation of caspase-3 after ST treatment were detected by western blotting analyses. The results in the present study are consistent with our previous results, which indicated that inducing apoptosis may be a common effect of ST in different cells in vitro.

Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

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.