Fusarium mycotoxin enniatin B: Cytotoxic effects and changes in gene expression profile.

The mycotoxin enniatin B, a cyclic hexadepsipeptide produced by the plant pathogen Fusarium, is prevalent in grains and grain-based products in different geographical areas. Although enniatins have not been associated with toxic outbreaks, they have caused toxicity in vitro in several cell lines. In this study, the cytotoxic effects of enniatin B were assessed in relation to cellular energy metabolism, cell proliferation, and the induction of apoptosis in Balb 3T3 and HepG2 cells. The mechanism of toxicity was examined by means of whole genome expression profiling of exposed rat primary hepatocytes. Enniatin B altered cellular energy metabolism and reduced cell proliferation in Balb 3T3 and HepG2 cell lines. Furthermore, the proportion of apoptotic cell populations of Balb 3T3 cells slightly increased. On the other hand, enniatin B caused necrotic cell death in primary hepatocytes. Gene expression studies revealed the alteration of energy metabolism due to effects on mitochondrial organization and function and the assembly of complex I of the electron transport chain.

The lager yeast Saccharomyces pastorianus removes and transforms Fusarium trichothecene mycotoxins during fermentation of brewer's wort.

An investigation was conducted to determine the fate of deoxynivalenol, deoxynivalenol-3-glucoside, HT-2 toxin and T-2 toxin, during a four-day fermentation with the lager yeast Saccharomyces pastorianus. The influence of excessive mycotoxin concentrations on yeast growth, productivity and viability were also assessed. Mycotoxins were dosed at varying concentrations to 11.5° Plato wort. Analysis of yeast revealed that presence of the toxins even at concentrations up to 10,000 µg/L had little or no effect on sugar utilisation, alcohol production, pH, yeast growth or cell viability. Of the dosed toxin amounts 9-34% were removed by the end of fermentation, due to physical binding and/or biotransformation by yeast. Deoxynivalenol-3-glucoside was not reverted to its toxic precursor during fermentation. Processing of full-scan liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) data with MetaboLynx and subsequent LC-QTOF-MS/MS measurements resulted in annotation of several putative metabolites. De(acetylation), glucosylation and sulfonation were the main metabolic pathways activated.

Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat.

To investigate the metabolic fate of HT-2 toxin (HT2) and T-2 toxin (T2) in wheat (Triticum aestivum L.), an untargeted metabolomics study utilizing stable isotopic labeling and liquid chromatography-high resolution mass spectrometry was performed. In total, 11 HT2 and 12 T2 derived in planta biotransformation products were annotated putatively. In addition to previously reported mono- and diglucosylated forms of HT2, evidence for the formation of HT2-malonyl-glucoside and feruloyl-T2, as well as acetylation and deacetylation products in wheat was obtained for the first time. To monitor the kinetics of metabolite formation, a time course experiment was conducted involving the Fusarium head blight susceptible variety Remus and the resistant cultivar CM-82036. Biotransformation reactions were observed already at the earliest tested time point (6 h after treatment), and formed metabolites showed different kinetic profiles. After ripening, less than 15% of the toxins added to the plants were determined to be unmetabolized.

Simultaneous determination of major type A and B trichothecenes, zearalenone and certain modified metabolites in Finnish cereal grains with a novel liquid chromatography-tandem mass spectrometric method.

A reliable and sensitive liquid chromatography-tandem mass spectrometric method was developed for the simultaneous quantitative determination in cereals of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol and zearalenone, as well as the modified metabolites 3-acetyl-deoxynivalenol, α-zearalenol, ß-zearalenol, deoxynivalenol-3-glucoside, HT-2-3-glucoside, nivalenol-3-glucoside, zearalenone-14-glucoside, zearalenone-14-sulphate, zearalenone-16-glucoside, α-zearalenol-14-glucoside and ß-zearalenol-14-glucoside. The 'dilute and shoot' approach was used for sample preparation after extraction with acetonitrile:water:acetic acid (79:20:1, v/v/v). Separation was carried out using reversed-phase liquid chromatography, and detection was performed using tandem mass spectrometry in the selected reaction monitoring mode. The method was in-house validated according to performance characteristics, established in Commission Regulation EC No 401/2006 and Commission Decision EC No 657/2002, prior to its application in a nationwide survey for the analysis of barley, oat and wheat samples (n = 95) harvested in Finland during 2013. Deoxynivalenol and its glucosylated form were the most abundant of the analytes, being detected in 93 and 81 % of the samples, respectively. Concentrations of deoxynivalenol were unusually high in 2013, especially in oats, with some cases exceeding the maximum legislative limits for unprocessed oats placed on the market for first-stage processing. All modified mycotoxins analysed were detected, and the natural occurrence of some of these compounds (e.g. zearalenone-16-glucoside and nivalenol-3-glucoside) in barley, oats and/or wheat was documented for the first time.

Repeated dose 28-day oral toxicity study of moniliformin in rats.

Moniliformin is a Fusarium mycotoxin mainly produced by several species infecting grains in different climatic conditions. According to our previous studies, it is acutely toxic to rats, with an LD50 cut-off value of 25mg/kg b.w. To further assess the possible health risks of low dose exposure to moniliformin, a subacute oral toxicity study was conducted in Sprague-Dawley rats, adapting OECD guideline 407. Five dose groups and two satellite groups, each consisting of five male rats, were daily exposed to moniliformin by gavage. Two rats in the highest dose group, showed decreased activity followed by acute heart failure and death. The rats of the lower doses (<9mg/kg b.w.) showed no signs of toxicity. The daily intake of moniliformin strongly reduced the phagocytic activity of neutrophils in all dose groups. The decrease continued in the satellite group during the follow-up period, indicating a severe impact on the immune system and a LOAEL value of 3mg/kg b.w. for moniliformin. Moniliformin was rapidly excreted into urine, ranging between 20.2 and 31.5% daily and showed no signs of accumulation. The concentration of moniliformin in faeces was less than 2%, which suggests efficient absorption from the gastrointestinal tract.

Determination of deoxynivalenol and deoxynivalenol-3-glucoside in wheat and barley using liquid chromatography coupled to mass spectrometry: on-line clean-up versus conventional sample preparation techniques.

In this study, we compared the performance of conventional sample preparation techniques used in mycotoxin analyses against automated on-line sample clean-up for the determination of deoxynivalenol (DON) and its conjugated derivative, deoxynivalenol-3-ß-d-glucoside (D3G), in cereal grains. Blank wheat and barley samples were spiked with DON and D3G, extracted with a mixture of acetonitrile:water (84:16, v/v) and processed by one of the following: extract and shoot, MycoSep(®) 227 clean-up columns, MycoSep 227 with an additional acetonitrile elution step and centrifugal filtration, followed by analysis with liquid chromatography tandem mass spectrometry. Based on method performance characteristics and poor recoveries (<30%) obtained for the polar D3G with some techniques, the extract and shoot approach was chosen for the inter-laboratory method comparison study. Thus, the same spiked samples were analysed in parallel by another laboratory with an in-house validated on-line sample clean-up method, utilising TurboFlow™ chromatography coupled to high resolution mass spectrometry. Method validation was performed by determination of specificity, linearity, recovery, intra-day precision and the limits of detection and quantification. Matrix-matched linearity (R(2)>0.985) was established in the range of 100-1600 and 20-320µg/kg for DON and D3G, respectively. Average recoveries (%RSD) were acceptable with both methods for wheat and barley, ranging between 73% and 102% (3-12%) for DON and 72% and 98% (1-10%) for D3G. The benefit of using automated sample clean-up in comparison to extract and shoot is the ability to inject directly pure extracts into the mass spectrometer, offering faster analyses and improved sensitivity with minimum system maintenance.

The intake of inorganic arsenic from long grain rice and rice-based baby food in Finland - low safety margin warrants follow up.

We evaluated total and inorganic arsenic levels in long grain rice and rice based baby foods on Finnish market. Inorganic arsenic was analysed with an HPLC-ICP-MS system. The total arsenic concentration was determined with an ICP-MS method. In this study, the inorganic arsenic levels in long grain rice varied from 0.09 to 0.28mg/kg (n=8) and the total arsenic levels from 0.11 to 0.65mg/kg. There was a good correlation between the total and inorganic arsenic levels in long grain rice at a confidence level of 95%. The total arsenic levels of rice-based baby foods were in the range 0.02 - 0.29mg/kg (n=10), however, the level of inorganic arsenic could only be quantitated in four samples, on average they were 0.11mg/kg. Our estimation of inorganic arsenic intake from long grain rice and rice-based baby food in Finland indicate that in every age group the intake is close to the lowest BMDL0.1 value 0.3µg/kg bw/day set by EFSA. According to our data, the intake of inorganic arsenic should be more extensively evaluated.

Trace level determination of polyether ionophores in feed.

A liquid chromatography-mass spectrometric method was developed and validated to determine six polyether ionophores (lasalocid sodium, monensin sodium, salinomycin sodium, narasin, maduramicin ammonium alpha, and semduramicin sodium) in feed samples. The method developed was very straightforward, involving extraction with 84% acetonitrile of the coccidiostats from the feed samples and filtration of the raw extract prior to chromatographic analysis. Method validation included the determination of selectivity, linearity, specificity, repeatability, the limit of detection, limit of quantification, decision limit (CC α ), detection capability (CC ß ), and recovery. Feed samples from the Finnish national feed control programme and suspected carry-over samples from a feed manufacturer were analysed in parallel with an existing liquid chromatography method coupled with ultraviolet detection. All feed control samples were negative in LC-UV, but with the developed MS method, monensin, salinomycin, and narasin were detected at concentrations of <0.025-0.73 mg/kg, <0.025-0.027 mg/kg, and <0.025-1.6 mg/kg, respectively. In suspected carry-over samples after an output of 2.0 tonnes of unmedicated feed in the pelletizer line, the concentrations of monensin, salinomycin, and narasin varied from undetected to 16 mg/kg. In the mixer line, after 3.2 tonnes of unmedicated feed output, the concentrations of monensin, salinomycin, and narasin varied from undetected to 2.4 mg/kg.

Application of OECD Guideline 423 in assessing the acute oral toxicity of moniliformin.

Moniliformin is a Fusarium mycotoxin highly prevalent in grains and grain-based products worldwide. In this study, the acute oral toxicity of moniliformin was assessed in Sprague-Dawley male rats according to OECD Guideline 423 with a single-dose exposure. Clinical observations and histopathological changes were recorded together with the excretion of moniliformin via urine and feces, utilizing a novel liquid chromatography-mass spectrometry method. According to our study, moniliformin is acutely toxic to rats with a rather narrow range of toxicity. Moniliformin can be classified into category 2 (LD(50) cut-off value 25 mg/kg b.w.), according to the Globally Harmonized System for the classification of chemicals. The clinical observations included muscular weakness, respiratory distress and heart muscle damage. Pathological findings confirmed that heart is the main target tissue of acute moniliformin toxicity. A significant proportion (about 38%) of the administered moniliformin was rapidly excreted in urine in less than 6 h. However, the toxicokinetics of the majority of the administered dose still requires clarification, as the total excretion was only close to 42%. Considering the worldwide occurrence of moniliformin together with its high acute toxicity, research into the subchronic toxicity is of vital importance to identify the possible risk in human/animal health.

Liquid chromatography tandem mass spectrometry determination of total budesonide levels in dog plasma after inhalation exposure.

A sensitive and selective method to quantify budesonide in dog plasma samples was developed and fully validated. Liquid-liquid extraction was followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry with electrospray ionization. After reconstitution of the analytes in the mobile phase, samples were analysed by reversed-phase liquid chromatography with isocratic elution. d8-Budesonide was used as an internal standard, and characteristic transitions of d8-budesonide and budesonide were used for quantification. The method was validated with respect to selectivity, specificity, linearity, recovery, repeatability, reproducibility and limits of detection and quantification. The validated method was successfully applied to monitor the plasma levels of budesonide in dogs exposed to clinical doses of inhaled and intravenous drug.

Analytical methods in DNA and protein adduct analysis.

DNA or protein adducts are reaction products of endogenous or exogenous chemicals and cellular macromolecules. Adducts are useful in toxicological studies and/or human biomonitoring exercises. In particular, DNA damage provides invaluable information for risk analysis. Second, metabolites or conjugates can be regarded as markers of phase II reactions though they may not give accurate information about the levels of reactive and damage-provoking reactive compounds or intermediates. Electrophiles are often short-lived molecules and therefore difficult to monitor. In contrast, adducts are often chemically stable, though their levels in biological samples are low, which makes their detection challenging. The assay of adducts is similar to the analysis of any other trace organic molecule, i.e. problems with the matrix and small amounts of analytes in samples. The (32)P-postlabelling assay is a specific method for DNA adducts but immunochemical and fluorescence-based methods have been developed which can detect adducts linked to both DNA and protein. Tandem mass spectrometry, particularly if combined with ultrahigh-performance liquid chromatography, is currently the recommended detection technique; however investigators are striving to develop novel ways to achieve greater sensitivity. Standards are a prerequisite in adduct analysis, but unfortunately they are seldom commercially available.

Exhaled pentane as a possible marker for survival and lipid peroxidation during radiotherapy for lung cancer--a pilot study.

To examine lipid peroxidation during radiotherapy (RT), exhaled pentane samples were collected from 11 lung cancer patients before RT and 30 and 120 min after the start of RT on days 1, 4 and 5 and at 30 and 40 Grays, if possible. Exhaled pentane samples were collected once from 30 healthy controls. Serum thiobarbituric-acid-reactive substances (TBARS) and conjugated dienes (CD) were obtained from patients on each exhaled air collection day. Lung cancer patients had higher exhaled pentane levels than controls (1.73 ng/L vs 0.83 ng/L, p=0.017). Exhaled pentane levels tended to decrease during the first RT day (p=0.075) and levels of CD decreased during the first week of RT (p=0.014). Higher pre-treatment pentane levels predicted better survival (p=0.003). Elevated exhaled pentane levels before RT may be due to the lipid peroxidation burden associated with cancer. The decrease of lipid peroxidation markers during RT may be attributable to enhanced antioxidant defense mechanisms.

Exposure to environmental tobacco smoke at work, at home, and during leisure time: a cross-sectional population sample.

Environmental tobacco smoke (ETS) is among the most common environmental health risks, with a striking and immediate biological response and increased disease risk. Exposure studies have looked mostly at worksite or home exposures, whereas total exposure levels at the population level are rarely reported. This study examined ETS exposure at work, at home, and during leisure time in a cross-sectional population sample of working-age adults. Our aim was to monitor changes in ETS exposure from 1992 to 2002. More detailed information on duration of exposure, distribution of exposure sites, and patterns of exposure was obtained in 2002. Data were based on Finland's national population chronic disease risk-factor surveys (conducted every 5 years). Total sample size varied from 8,000 to 13,500. The survey includes a self-administered questionnaire about ETS exposure at different sites. The proportion of nonsmoking persons exposed to ETS declined throughout the study period among both men and women. In 2002, 5.9% of male and 3.6% of female nonsmokers were exposed to ETS 1 hour or more per day, whereas 5.8% of men and 1.7% women were exposed less than 1 hour daily. Worksite exposure was more common among younger age groups of both sexes, but nonsmoking women in older age groups received more exposure at home than at worksites. Policy developments on ETS should aim to protect the whole population from ETS in all environments given that health risks from ETS often persist at home and in leisure environments. Total exposure levels should be studied to assess the health impacts of ETS.

Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry.

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.

An integrated sample preparation to determine coccidiostats and emerging Fusarium-mycotoxins in various poultry tissues with LC-MS/MS.

The usefulness of an existing sample preparation technique used for ionophoric coccidiostats (lasalocid, monensin, salinomycin and narasin) was applied in the analysis of emerging Fusarium-mycotoxins beauvericin (BEA) and enniatins (ENNs) in poultry tissues (liver and meat). Also, maduramicin and liver as a new sample matrix was introduced. The developed methods were validated and applied for the determination of coccidiostats and BEA/ENNs in Finnish poultry tissues in 2004-2005. The validation parameters demonstrated that the integrated sample preparation technique is applicable to the parallel determination of these contaminants in poultry tissues. Of the samples analysed (276 meat and 43 liver), only trace levels of LAS, MON, SAL, NAR and MAD were detected in 7, 3, 5, 6 and 4% of the samples, respectively. Interestingly, for the first time, traces of BEA and ENNs could also be detected in animal tissues. BEA and ENNs A, A1, B and B1 were found in 2, 0.3, 0.6, 4 and 3% of the samples, respectively. The simultaneous presence of coccidiostats and mycotoxins was detected in three turkey samples in 2004.

Acrylamide levels in Finnish foodstuffs analysed with liquid chromatography tandem mass spectrometry.

Sample clean-up and HPLC with tandem mass spectrometric detection (LC-MS/MS) was validated for the routine analysis of acrylamide in various foodstuffs. The method used proved to be reliable and the detection limit for routine monitoring was sensitive enough for foods and drinks (38 microg/kg for foods and 5 microg/L for drinks). The RSDs for repeatability and day-to-day variation were below 15% in all food matrices. Two hundred and one samples which included more than 30 different types of food and foods manufactured and prepared in various ways were analysed. The main types of food analysed were potato and cereal-based foods, processed foods (pizza, minced beef meat, meat balls, chicken nuggets, potato-ham casserole and fried bacon) and coffee. Acrylamide was detected at levels, ranging from nondetectable to 1480 microg/kg level in solid food, with crisp bread exhibiting the highest levels. In drinks, the highest value (29 microg/L) was found in regular coffee drinks.

A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry.

A quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile-acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC-MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCalpha) for MG and LMG was 0.13 and 0.16 microgkg(-1). The respective detection capabilities (CCbeta) were 0.22 and 0.27 microgkg(-1). The absolute recovery (repeatability SD(r)) was in the range of 58-65% (7.8-11.2%) for MG and 59-68% (9.7-16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SD(r)) of 103-110% (4.8-9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.

Simultaneous determination of four coccidiostats in eggs and broiler meat: validation of an LC-MS/MS method.

A published confirmatory method for the quantitative determination of four ionophoric coccidiostats (lasalocid, monensin, salinomycin and narasin) in eggs and broiler meat has been further developed. It is proposed for replacement of liquid chromatography methods previously used in analysis of ionophoric coccidiostats. The samples were extracted with acetonitrile and purified on a silica solid phase extraction column. Purified samples were analysed by liquid chromatography-mass spectrometry and the method, was validated according to the Commission Decision 2002/657/EC. The validation parameters selectivity, linearity, specificity, precision, recovery, decision limit (CCalpha) and detection capability (CCbeta) were determined. The recoveries of coccidiostats analysed ranged from 64-99% in eggs and 62-100% in broiler meat. CCalpha varied from 0.8-1.4 microg/kg in eggs and from 1.5-2.5 microg/kg in broiler meat. CCbeta varied from 0.9 microg/kg to 2.0 microg/kg in eggs and from 1.7-3.2 microg/kg in broiler meat.

Inhalation exposure to 1,3-butadiene and styrene in styrene-butadiene copolymer production.

This study assessed personal exposure to 1,3-butadiene (BD) and styrene in three plants manufacturing styrene-butadiene (SB) copolymers. Air samples were collected from the breathing zone of 28 workers over 4 months in three SB plants using diffusive samplers. The total number of samples was 885 with the number of samples per participant varying from 19 to 39. Samples were collected by use of 3M 3500 passive monitors and analyzed with a gas chromatograph (GC). Sampling proved to be simple and inexpensive and laboratory analysis of BD could detect 0.01 and 0.007 part per millions (ppm) of styrene in the 8h samples. In the case of BD, 624 samples were below the limit of quantification (LOQ), 240 samples were between the LOQ and 1 ppm, and 21 samples exceeded the threshold limit value (TLV). In the case of styrene 336 samples were below the LOQ, 548 samples were between the LOQ and 20 ppm. The TLV was exceeded once. The data gives a comprehensive picture of personal exposure of workers in modern SB latex manufacturing plants. The study illustrates also how the new TLV of BD is being implemented.

The residue levels of narasin in eggs of laying hens fed with unmedicated and medicated feed.

Laying hens were fed contaminated feed containing narasin 2.5 mg/kg for 21 days followed by a 7 day withdrawal period, hens in the control group were fed unmedicated feed. Eggs were collected during trial days 0, 3, 7, 14, 21 and after the withdrawal period of 7 days. The concentration of narasin in yolks and egg whites was analyzed by a liquid chromatography-mass spectrometry method. Narasin was found to accumulate in yolks, where the narasin concentration increased during the treatment. The concentration of narasin varied from 5.9 to 13.8 microg/kg (mean 10.6 microg/kg) in yolks after 21 day feeding periods. The concentrations of narasin ranged from < 0.9 to 1.4 microg/kg after the withdrawal period. Narasin residues were not found in egg whites of the laying hens fed contaminated feed nor in either yolks or egg whites of the laying hens fed unmedicated feed. The effect of cooking was also tested on the amount of narasin residues in eggs. Cooking for 10 min did not significantly influence the narasin residues in eggs. Traces of lasalocid were also found in the yolks. The traces of lasalocid are attributable to an accidental contamination of the feed during its manufacture.