Computational methods meet in vitro techniques: A case study on fusaric acid and its possible detoxification through cytochrome P450 enzymes.

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.

Potential toxicity assessment of mycotoxin fusaric acid with the spectral shift profile on DNA.

In this study, the multiple toxicities induced by three different doses (1, 5, and 10 µM) of fusaric acid (FA), a mycotoxin, was investigated with Allium test. Physiological (percent germination, root number, root length, and weight gain), cytogenetic (micronucleus = MN, chromosomal abnormalities = CAs, and mitotic index = MI), biochemical (proline level, malondialdehyde = MDA level, catalase = CAT activity, and superoxide dismutase = SOD activity), and anatomical parameters were used as indicators of toxicity. Allium cepa L. bulbs were divided into four groups as one control and three applications. The bulbs in the control group were germinated with tap water for 7 days, and the bulbs in the treatment groups were germinated with three different doses of FA for 7 days. As a result, FA exposure caused a decrease in all physiological parameters examined at all three doses. Besides, all FA doses caused a decrease in MI and an increase in the frequency of MN and the number of CAs. FA promoted CAs such as nucleus with vacuoles, nucleus buds, irregular mitosis, bridge, and misdirection in root meristem cells. DNA and FA interactions, which are the possible causes of genotoxic effects, were examined by spectral analysis, and FA could interact with DNA through intercalation, causing bathochromic and hypochromic shifts in the spectrum. FA also causes toxicity by inducing oxidative stress in cells, confirming this; dose-related increases in root MDA and proline levels were measured as a result of FA exposure. In the root SOD and CAT enzyme activities, increases up to 5 µM doses and decreases at 10 µM doses were measured. FA exposure induced anatomical damage such as necrosis, epidermis cell damage, flattened cell nucleus, thickening of the cortex cell wall, and unclear vascular tissue in root tip meristem cells. As a result, FA caused a comprehensive toxicity by showing an inhibitory effect in A. cepa test material, and the Allium test was a very useful test in determining this toxicity.

Potential of Burkholderia sp. IMCC1007 as a biodetoxification agent in mycotoxin biotransformation evaluated by mass spectrometry and phytotoxicity analysis.

Microbial degradation is considered as an attractive method to eliminate exposure to mycotoxin that cause a serious threat in agriculture global industry and severe human health problems. Compared with other more prominent mycotoxin compounds, fusaric acid (FA) biodegradation has not been widely investigated. In this study, a fusaric acid-degrading bacterium Burkholderia sp. IMCC1007 was identified by 16 S rRNA gene sequencing and its detoxification characteristics were evaluated. This strain able to utilize FA as sole energy and carbon source with growth rate (µ) of 0.18 h- 1. Approximately 93% from the initial substrate FA concentration was almost degraded to the residual about 4.87 mg L- 1 after 12 h of incubation. The optimal degradation conditions for pH and temperature were recorded at 6.0 with 30 °C respectively. An efficient FA degradation of strain IMCC1007 suggested its potential significance to detoxification development. Accroding to LC-MS/Q-TOF analysis, FA was bio-transformed to 4-hydroxybenzoic acid (C7H6O3) and other possible metabolites. Plant treated with detoxified FA products exhibited reduction of wilting index, mitigating against FA phytoxicity effect on plant growth and photosynthesis activity. Phytotoxicity bioassay suggested that degradation product of IMCC1007 was not a potent harmful compound towards plants as compared to the parent compound, FA. As a conslusion, our study provides a new insight into the practical application of biodetoxifcation agent in controlling mycotoxin contamination.

A target fishing study to spot possible biological targets of fusaric acid: Inhibition of protein kinase-A and insights on the underpinning mechanisms.

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.

Zinc and Copper Enhance Cucumber Tolerance to Fusaric Acid by Mediating Its Distribution and Toxicity and Modifying the Antioxidant System.

Fusaric acid (FA), the fungal toxin produced by Fusarium oxysporum, plays a predominant role in the virulence and symptom development of Fusarium wilt disease. As mineral nutrients can be protective agents against Fusarium wilt, hydroponic experiments employing zinc (Zn) and copper (Cu) followed by FA treatment were conducted in a glasshouse. FA exhibited strong phytotoxicity on cucumber plants, which was reversed by the addition of Zn or Cu. Thus, Zn or Cu dramatically reduced the wilt index, alleviated the leaf or root cell membrane injury and mitigated against the FA inhibition of plant growth and photosynthesis. Cucumber plants grown with Zn exhibited decreased FA transportation to shoots and a 17% increase in toxicity mitigation and showed minimal hydrogen peroxide, lipid peroxidation level with the increased of antioxidant enzymes activity in both roots and leaves. Cucumber grown with additional Cu absorbed less FA but showed more toxicity mitigation at 20% compared to with additional Zn and exhibited decreased hydrogen peroxide level and increased antioxidant enzymes activity. Thus, adding Zn or Cu can decrease the toxicity of the FA by affecting the absorption or transportation of the FA in plants and mitigate toxicity possibly through chelation. Zn and Cu modify the antioxidant system to scavenge hydrogen peroxide for suppressing FA induction of oxidative damage. Our experiments could provide a theoretical basis for the direct application of micro-fertilizer as protective agents in farming.

The neglected foodborne mycotoxin Fusaric acid induces bioenergetic adaptations by switching energy metabolism from mitochondrial processes to glycolysis in a human liver (HepG2) cell line.

Metabolic flexibility defines the capacity of cells to respond to changes in nutrient status. Mitochondria are important mediators of metabolic flexibility and dysfunction is associated with metabolic inflexibility and pathology. Foodborne toxins are often overlooked as potential factors contributing to metabolic toxicity. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial function. The aim of this study was to investigate the molecular mechanisms underlying a metabolic switch in response to FA. This study investigated the effects of FA on energy homeostasis in cultured human liver (HepG2) cells. HepG2 cells poised to undergo oxidative and glycolytic metabolism were exposed to a range of FA concentrations (4, 63 and 250 µg/mL) for 6 h. We determined mitochondrial toxicity, acetyl CoA levels and cell viability using luminometric, fluorometric and spectrophotometric methods. Expression of metabolic proteins (PDK1, PKM2, phosphorylated-PDH E1α and HIF-1α) and mRNAs (HIF-1α, PKM2, LDHa and PDK1) were determined using western blot and qPCR respectively. Our data connects a constitutive expression of HIF-1α in response to FA, to the inhibition of pyruvate decarboxylation through up-regulation of PDK-1 and phosphorylation of Pyruvate Dehydrogenase E1α subunit. Moreover, we highlight the potential of FA to induce a glucose "addiction" and phenotype reminiscent of the Warburg effect. The findings provide novel insights into the impact of this neglected foodborne mycotoxin in the dysregulation of energy metabolism.

Evaluation of the cytotoxic and genotoxic effects of mycotoxin fusaric acid.

Fusaric acid (FA) is produced by several Fusarium species and is commonly found in grains. This investigation was performed to evaluate the cytotoxic and genotoxic effects of FA either in human cervix carcinoma (HeLa) cell line using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay and in human lymphocytes using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) as well as comet assay in vitro. The cells were treated with 0.78, 1.56, 3.125, 6.25, 12.50, 25, 50, 100, 200, and 400 µg/mL concentrations of FA. It has potent cytotoxic effect on HeLa cell line measured by MTT assay especially at higher concentrations (200, 400 µg/mL). The half of inhibitory concentration (IC50) evidenced by FA in the HeLa cells was 200 µg/mL at 24 h and between 200 and 400 µg/mL at 48 h. It was also observed that FA produced a significant decrease in mitotic index (MI) at 12.50 µg/mL compared to solvent control. Furthermore, it indicated a cytotoxic effect at the concentrations ranging from 25 to 400 µg/mL in human lymphocytes. The results of this research point out that being exposed to FA at high concentrations show cytotoxicity. Besides FA induced comet tail intensity at 3.125, 6.25, and 12.50 µg/mL concentrations in isolated human lymphocytes. On the other hand, no genotoxic effects were seen in human lymphocytes in vitro using CA, SCE and MN assays.

Molecular docking and mechanisms of fusaric acid induced mitochondrial sirtuin aberrations in glycolytically and oxidatively poised human hepatocellular carcinoma (HepG2) cells.

Fusaric acid (FA) is a ubiquitous yet neglected mycotoxin. The toxicity of FA is associated with mitochondrial dysfunction and oxidative stress. Sirtuins (SIRTs) are key mediators of cell stress responses through deacetylation of antioxidant, mitochondrial maintenance and energy metabolism proteins. Dietary bioactive compounds have profound effects on SIRT activity, however little is known regarding common foodborne toxins and SIRTs. In this study the interaction of FA with mitochondrial SIRTs - SIRT3 and SIRT5, were firstly studied by molecular docking. Thereafter we substantiated the in silico findings by investigating the effect of FA on expression profiles of SIRT3 and SIRT5, and transcriptional and post-transcriptional regulators, PGC-1α and miRNA-30c using western blots and qPCR in vitro. FA was predicted to bind to the active site of SIRT3 and SIRT5 having implications for biological activity. Furthermore, protein expression of SIRT3 and SIRT5 was down-regulated despite elevated mRNA levels. Further experimentation revealed post-transcriptional regulation of both SIRTs as evidenced by elevated miRNA-30c despite induction of PGC-1α. This study highlights the potential of a diet contaminated with FA to dysregulate mitochondrial specific proteins that can lead to initiation and progression of sirtuin related diseases including cancer and insulin resistance.

Dose-dependent neuroprotective effect of oriental phyto-derived glycyrrhizin on experimental neuroterminal norepinephrine depletion in a rat brain model.

The dose-dependent neuroprotective role of licorice-derived glycyrrhizin during subacute neuroterminal norepinephrine (NE) depletion was studied in rat brain. Experimental design included thirty 5-week-old male rats randomly divided into five groups. Compared to the saline-injected control group, the group receiving daily intraperitoneal injection of fusaric acid (FA; 5 mg/kg/b.w.) for 30 days showed pharmacological depletion of NE. The neuroprotective effects of three successively increasing oral doses of glycyrrhizin were examined in FA-treated rats. Neurochemical parameters and histo-/immunohistopathological changes in the hippocampus were examined. FA generated global hippocampal stress with altered neurobiochemical parameters, accompanied by immune-confirmed inflammatory tissue damage, and noticeable behavioral changes. Although glycyrrhizin after FA-induced intoxication did not correct the recorded drop in the NE level, it decreased the dopamine levels to control levels. Similarly, glycyrrhizin at a high dose restored the serotonin level to its normal value and blocked the FA-induced increase in the level of its metabolite, 5-hydroxyindoleacetic acid. The FA-induced rise in γ-aminobutyric acid (GABA) and histamine was alleviated after administration of a high dose of glycyrrhizin. This was accompanied by improvements in the bioenergetic status and neuronal regenerative capacity through recovery of ATP and brain-derived neurotrophic factor levels to the pre-intoxicated values. High doses of glycyrrhizin also ameliorated the FA-generated behavioral changes and oxidative damage, manifested by the reduction in the expression of cortical pro-apoptotic caspase 3 in the same group. This study suggests that glycyrrhizin can potentially mend most of the previously evoked neuronal damage induced by FA intoxication in the brain of an experimental rat model.

Role of fusaric acid in the development of 'Fusarium wilt' symptoms in tomato: Physiological, biochemical and proteomic perspectives.

Fusarium wilt is one of the most prevalent and damaging diseases of tomato. Among various toxins secreted by the Fusarium oxysporum f. sp. lycopersici (causal agent of Fusarium wilt of tomato), fusaric acid (FA) is suspected to be a potent pathogenicity factor in tomato wilt disease development. With this rationale the present study was carried out with physiological, biochemical and proteomic perspectives. Treatment of FA was given to the leaves of tomato directly through infiltration to show the characteristic features of Fusarium wilt of tomato. The phytotoxic effect of FA was assessed in the form of cell death in tomato leaves which was observed by increased uptake of Evans blue stain. The measurement of electrolyte leakage was used as an indicator of the extent of cell death. The influence of FA on the leaf photosynthesis of tomato plant was investigated and it was found that FA strongly reduced the photosynthetic pigment contents of tomato leaves resulting to heavy suppression of leaf photosynthesis processes, which therefore affected leaf physiology finally leading to leaf wilting and necrosis. This cell death inducer (FA) produced an enormous oxidative burst during which large quantities of reactive oxygen species (ROS) like H2O2 was generated in the treated leaf tissues of tomato plants which was evident from enhancement in lipid peroxidation. To assess the involvement of proteolysis in the cell death cascade induced by FA treatment, total protease activity was measured in the leaf tissues and it was found that the total protease activity increased with the treatment and leading to cell death. Furthermore, proteomic study was used as a powerful tool to understand the alterations in cellular protein expression in response to FA exposure. Differential expression in several proteins was observed in the present study. Proteomic analyses, thus, clearly indicate that proteins belonging to different functional classes are significantly affected in the plant leaf tissues after FA exposure leading to deterioration of structure and metabolism of cells. Thus, it is concluded that FA plays an important role in fungal pathogenicity by decreasing cell viability.

Fusaric Acid immunotoxicity and MAPK activation in normal peripheral blood mononuclear cells and Thp-1 cells.

Fusaric acid (FA), a food-borne mycotoxin, is a potent divalent metal chelator. The human immune system is complex and susceptible to environmental insult however, the immunotoxity of FA remains unknown. We investigated the immunotoxicity of FA on human peripheral blood mononuclear cells (PBMCs) and Thp-1 cells. FA was cytotoxic to PBMCs (IC50-240.8 µg/ml) and Thp-1 (IC50-107.7 µg/ml) cells at 24 h. FA induced early apoptosis but significantly decreased caspase activity in PBMCs, a characteristic of paraptosis. In Thp-1 cells, FA induced apoptosis and increased caspase -9 and -3/7 activities. In PBMCs, FA maintained mitochondrial membrane potential and decreased protein expression of Bax whilst increasing expression of p-Bcl-2; FA induced oxidative stress and depleted ATP levels in both cell types. In Thp-1 cells, FA increased mitochondrial membrane depolarization and decreased p-Bcl-2 expression. In PBMCs, FA significantly up-regulated the MAPK protein expression of p-ERK and p-JNK but down-regulated p-p38 expression. In Thp-1 cells, FA up-regulated MAPK protein expression of p-ERK whilst p-JNK and p-p38 expression were down-regulated. In conclusion FA induced programmed cell death and altered MAPK signaling in healthy PBMCs and Thp-1 cells strongly suggesting a possible mechanism of FA induced immunotoxicity in vitro.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.

Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

New Fusaric Acid Derivatives from the Endophytic Fungus Fusarium oxysporum and Their Phytotoxicity to Barley Leaves.

Chemical investigation of the endophytic fungus Fusarium oxysporum isolated from fruits of Drepanocarpus lunatus afforded eight new fusaric acid derivatives, fusaricates A-G, 1-7, and 10-hydroxy-11-chlorofusaric acid, 8, along with four known compounds. Their structures were elucidated by one- and two-dimensional NMR as well as MS data and by comparison with the literature. The absolute configurations of fusaricates C-E, 3-5, were determined using chiral GC-MS. Fusaricates A-G, 1-7, represent the first examples of fusaric acid linked to a polyalcohol moiety via an ester bond. All isolated fusaric acid derivatives 1-8 showed significant phytotoxicity to leaves of barley.

Production of siderophores increases resistance to fusaric acid in Pseudomonas protegens Pf-5.

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe2+ and Fe3+, but also Zn2+, Mn2+ and Cu2+, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.

Conversion of fusaric acid to Fusarinol by Aspergillus tubingensis: a detoxification reaction.

The fungus Fusarium oxysporum causes wilt diseases of plants and produces a potent phytotoxin fusaric acid (FA), which is also toxic to many microorganisms. An Aspergillus tubingensis strain with high tolerance to FA was isolated from soil and designated as CDRAt01. HPLC analysis of culture filtrates from A. tubingensis isolate CDRAt01 grown with the addition of FA indicated the formation of a metabolite over time that was associated with a decrease of FA. Spectral analysis and chemical synthesis confirmed the compound as 5-butyl-2-pyridinemethanol, referred to here as fusarinol. The phytotoxicity of fusarinol compared to FA was measured by comparing necrosis induced in cotton (Gossypium hirsutum L. cv. Coker 312) cotyledons. Fusarinol was significantly less phytotoxic than FA. Therefore, the A. tubingensis strain provides a novel detoxification mechanism against FA which may be utilized to control Fusarium wilt.

Fusaric acid accelerates the senescence of leaf in banana when infected by Fusarium.

Fusarium oxysporum f.sp. cubense (FOC) is a causal agent of vascular wilt and leaf chlorosis of banana plants. Chloroses resulting from FOC occur first in the lowest leaves of banana seedlings and gradually progress upward. To investigate the responses of different leaf positions to FOC infection, hydroponic experiments with FOC inoculation were conducted in a greenhouse. Fusarium-infected seedlings exhibited a decrease in net photosynthesis rate, stomatal conductance, and transpiration rate of all leaves. The wilting process in Fusarium-infected seedlings varied with leaf position. Measurements of the maximum photochemical efficiency of photosystem II (F(V)/F(max) and visualization with transmission electron microscopy showed a positive correlation between chloroplast impairment and severity of disease symptoms. Furthermore, results of malondialdehyde content and relative membrane conductivity measurements demonstrated that the membrane system was damaged in infected leaves. Additionally, the activities of phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were increased and total soluble phenolic compounds were significantly accumulated in the leaves of infected plants. The structural and biochemical changes of infected plants was consistent with plant senescence. As the FOC was not detected in infected leaves, we proposed that the chloroplast and membrane could be damaged by fusaric acid produced by Fusarium. During the infection, fusaric acid was first accumulated in the lower leaves and water-soluble substances in the lower leaves could dramatically enhance fusaric acid production. Taken together, the senescence of infected banana plants was induced by Fusarium infection with fusaric acid production and the composition of different leaf positions largely contribute to the particular senescence process.

Effects of fusaric acid treatment on HEp2 and docetaxel-resistant HEp2 laryngeal squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) can become resistant to some chemotherapy and becomes a clinical challenge when it recurs. There are few agents identified for second-line treatment of resistant head and neck cancer. Therefore, we evaluated the role of fusaric acid (FA) as a possible agent for treatment for chemotherapy-resistant laryngeal squamous cell carcinoma. METHODS: The HEp2 and docetaxel-resistant HEp2 (HEp-Doc) cell lines were created from human laryngeal cancer. Cell lines were exposed to FA at increasing concentrations (0.1, 0.3, and 0.5 mM) and time intervals of 24, 48, 72, and 96 h. The effects on cell survival, apoptosis, and cytokine and protein expression were analyzed. RESULTS: FA treatment in the HEp-Doc cells showed greater reduction of cell number and colony-forming units and more apoptosis when compared to HEp2 and required less time to reduce cell number. Four cytokines were detected in HEp2 cancer cells that increased with FA treatment. Pro-caspase-9 and -7 and poly (ADP-ribose) polymerase also increased with FA treatment. CONCLUSION: FA may be an agent to be considered in cases of HNSCC treatment resistance or post-docetaxel recurrence. Further investigation of FA in vitro and in vivo is indicated.

Phytotoxicity of fusaric acid and analogs to cotton.

We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 µL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming.

Feed contaminated with Fusarium toxins alter lymphocyte proliferation and apoptosis in primiparous sows during the perinatal period.

Two groups of pregnant primiparous sows (day 89 ± 2 of gestation) were 54 days (± 1 day) fed either with an experimental diet (5.08 mg kg(-1) deoxynivalenol--DON, 0.09 mg kg(-1) zearalenone and 21.61 mg kg(-1) fusaric acid) or control diet (0.25 mg kg(-1) DON). The consummation of feed was significantly higher in the control group. Lymphocyte stimulation in culture from peripheral blood lymphocytes (PBL) was measured by BrdU incorporation test using two different concentrations of mitogen PHA 10 and 20 µg ml(-1), two different concentrations of DON (5 and 1 µg ml(-1)) and a combination of both, PHA and DON (PHA 10+DON 5, PHA 10+DON 1 and PHA 10+DON 0.1 µg ml(-1)). The lymphocyte proliferation, except for PHA 10 µg ml(-1), was significantly higher in the experimental group. Further on, using the photometric enzyme immunoassay, the apoptosis was studied in non-stimulated 72h lymphocyte culture or stimulated with 1 µg ml(-1) of DON. The significantly higher apoptosis was in non-stimulated lymphocyte cultures from the experimental group (P = 0.036). The results suggest that such feed may affect the peripheral lymphocyte population in the direction of their proliferation response and programmed cell death.