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.

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.

Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins.

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe.

Deleterious effects of mycotoxin combinations involving ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with carcinogenic properties. Its presence was detected in various foodstuffs all over the world but with significantly higher frequency and concentrations in areas with endemic nephropathy (EN). Even though food is often contaminated with more than one mycotoxin, earlier studies focused on the occurrence and toxicology of only OTA. Only a limited number of surveys showed that OTA co-occurs in food with mycotoxins (citrinin-CIT, penicilic acid, fumonisin B1-FB1, aflatoxins-AF) which exert nephrotoxic, carcinogenic or carcinogen-promoting activity. This review summarises the findings on OTA and its co-occurrence with the mentioned mycotoxins in food as well as experimental data on their combined toxicity. Most of the tested mycotoxin mixtures involving OTA produced additive or synergistic effects in experimental models suggesting that these combinations represent a significant health hazard. Special attention should be given to mixtures that include carcinogenic and cancer-promoting mycotoxins.

Occupational exposure to airborne fungi in two Croatian sawmills and atopy in exposed workers.

Airborne fungi were collected over a one year period at 2-month intervals at 2 sawmills in Croatia (SM 1 and SM 2) processing mainly beech wood and oak wood. A questionnaire concerning respiratory symptoms and skin prick test (SPT) with common inhalatory allergens and moulds Cladosporium herbarum, Alternaria alternata, Aspergillus niger, Penicillium notatum, and Rhizopus nigricans were performed in 96 workers from the same sawmills. Average concentrations of airborne fungi were 1,696-7,316 cfu/m(3) in SM 1 and 1,706-4,819 cfu/m(3) in SM 2, respectively. Health hazardous levels of airborne fungi (above 10 4 /m(3)) were present only in SM 1. These levels were related to saw working sites and were season-dependent, i.e. present only during the summer. Penicillium (50-100%), Paecilomyces (43-100%) and Chrysonilia (33-100%) dominated among 17 fungal genera identified in both sawmills. Symptoms of rhinitis, asthma, and dry cough were most frequently recorded among analysed workers. SPT to moulds was negative in all tested workers, except one positive to R. nigricans, indicating that moderate airborne fungi levels found in the analysed sawmills were not related to IgE-mediated sensitization to moulds in exposed workers, even in atopics. Atopy was present among woodworkers in similar proportions to the general population of Croatia, suggesting that the wood-processing industry is not selective for atopic workers.

Cytotoxicity and genotoxicity of versicolorins and 5-methoxysterigmatocystin in A549 cells.

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.

A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin.

The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24 h) demonstrated that CTN (IC(50) = 73.5 ± 1.0, 75.4 ± 1.4 µM, respectively) was less toxic than OTA (IC(50) = 14.0 ± 2.4, 20.5 ± 1.0 µM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10 µM) and CTN (30 and 50 µM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12 h than 24 h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24 h than 12 h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTA + CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTA + CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTA + CTN. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.

Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action.

This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC(50) of BEA (5.0 +/- 0.6) and OTA (15.8 +/- 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 microM) and OTA (1 and 5 microM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 muM of OTA alone and BEA + OTA combinations. Single BEA (0.5 microM) and OTA (1 and 5 microM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans.

«Suspects¼ in etiology of endemic nephropathy: aristolochic acid versus mycotoxins.

Despite many hypotheses that have been challenged, the etiology of endemic nephropathy (EN) is still unknown. At present, the implications of aristolochic acid (AA) and mycotoxins (ochratoxin A-OTA and citrinin-CIT) are under debate. AA-theory is based on renal pathohistological similarities between Chinese herbs nephropathy (CHN) and EN, findings of AA-DNA adducts in EN and in patients with urinary tract tumors (UTT), as well as the domination of A:T®T:A transversions in the p53 mutational spectrum of UTT patients, which corresponds with findings of such mutations in AA-treated rats. However, exposure pathways of EN residents to AA are unclear. Experimental studies attempting to deduce whether nephrotoxins OTA and CIT appear at higher frequencies or levels (or both) in the food and blood or urine of EN residents support the mycotoxin theory. Also, some molecular studies revealed the presence of OTA-DNA adducts in the renal tissue of EN and UTT patients. In this review, data supporting or arguing against AA and mycotoxin theory are presented and discussed.

Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzyme-linked immunosorbent assay and thin-layer chromatography.

Aspergillus, Penicillium, and Fusarium species frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 microg kg-1), followed by FBs (27%, 3690 microg kg-1), AFs (24.3%, 4.6 microg kg-1), and OTA (16.2%, 9.8 microg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases.

Cytotoxicity and apoptosis induced by fumonisin B(1), beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment.

The objective of this study was to determine individual and combined effects of fumonisin B(1) (FB(1)), beauvericin (BEA) and ochratoxin A (OTA) on porcine kidney epithelial PK15 cell survival by measuring lactate dehydrogenase (LDH) activity, apoptotic index and caspase-3 activity. Cells were treated with 0.05, 0.5 and 5 microg/ml of each mycotoxin or with the combinations of two or all three mycotoxins for 24 and 48 h. Changes in LDH and caspase-3 activity, and in apoptotic index showed that the cytotoxic and apoptotic effects of these mycotoxins were concentration- and time- dependent. Significant increase of LDH activity was observed after 48 h of exposure to the highest concentration of FB(1) (45%), BEA (84%) and OTA (77%), as compared to control. OTA increased caspase-3 activity after 24 h of treatment with 0.5 mug/mL (84%), while BEA (319%) and FB(1) (419%) significantly affected this enzyme activity after 48 h (P < 0.05). Increase of caspase-3 activity preceded significant morphological apoptotic changes, which were detected after 48 h of exposure to a single toxin. Combined treatment with FB(1), BEA and OTA resulted mostly in additive effects on LDH activity, and additive and synergistic effects on caspase-3 activity and apoptotic index.

Lipid peroxidation and glutathione levels in porcine kidney PK15 cells after individual and combined treatment with fumonisin B(1), beauvericin and ochratoxin A.

Individual and combined effects of the mycotoxins fumonisin B(1), beauvericin and ochratoxin A on cell viability, lipid peroxidation (TBARS) and intracellular glutathione (GSH) were studied on porcine kidney epithelial cells (PK15). Cells were treated with 0.05, 0.5 and 5 microg/ml of each mycotoxin or the combinations of two or all three applied in equal concentrations for 24 and 48 hr. Changes in cell viability, GSH and TBARS levels showed that the cytotoxic effects of these mycotoxins were concentration- and time-dependent. After 24 hr, cell viability was significantly decreased by the exposure to 5 microg/ml of fumonisin B(1) (25%), beauvericin (30%) and ochratoxin A (35%), as compared to controls. Only ochratoxin A (5 microg/ml) increased TBARS (56%), with further significant increase (85%) after 48 hr exposure. Fumonisin B(1) and beauvericin significantly increased TBARS (57% and 80%, respectively) only when the highest dose was applied for 48 hr. After 24 hr, GSH was significantly decreased (18%) by ochratoxin A (0.05 microg/ml), whereas fumonisin B(1) and beauvericin significantly decreased GSH at the concentration of 0.5 microg/ml. Combined treatment with fumonisin B(1), beauvericin and ochratoxin A resulted mostly in additive effects especially after a 24-hr exposure, although synergistic as well as antagonistic interactions could not be excluded depending on toxin concentrations and time of exposure. This is the first report on beauvericin-induced effects on lipid peroxidation and GSH in animal cells.

[Beauvericin: chemical and biological aspects and occurrence]. / Bovericin: kemizam, bioloski aspekti i rasirenost.

Beauvericin (BEA) is a cyclic hexadepsipeptide produced by Beauveria bassiana, Paecilomyces fumosoroseus, Paecilomyces tenuipes, Polyporus sulphurous, and a variety of Fusarium species. This mycotoxin shows antimicrobial, insecticidal, cytotoxic, and apoptotic activity. It is the most potent specific inhibitor of cholesterol acyltransferase and possesses ionophoric properties. BEA increases ion permeability in biological membranes by forming a complex with essential cations (Ca2+, Na+, K+), which may affect the ionic homeostasis. BEA has been frequently found in maize samples in Europe, USA and Africa and co-contamination with other Fusarium toxins such as fumonisins, and moniliformin was also found. There is only one report of BEA occurrence and co-occurrence with fumonisin B1, fumonisin B2 and ochratoxin A in Croatia. Biological activity of BEA may increase the toxicity of other mycotoxins that co-occur with BEA in food. The role of BEA in the development of human and animal mycotoxicosis is still unknown.