Tadahiro Suzuki

Gene expression profiles of yeast Saccharomyces cerevisiae sod1 caused by patulin toxicity and evaluation of recovery potential of ascorbic acid.

Patulin (PAT) is a fungal secondary metabolite and exhibits various toxicities including DNA damage and oxidative stress. These toxicities are eased by ascorbic acid (AsA). Although a number of studies regarding the mitigating effect of AsA against PAT toxicity have been reported, a comprehensive study about gene expressions is currently underway. Here, we carried out a detailed evaluation of PAT toxicity by co-incubation with AsA using the superoxide dismutase (SOD) mutant. DNA microarray results extracted the alterations in iron transporter and Fe/S cluster assembly genes; some of the genes that constitute the cellular iron transporter systems remained dysfunctional even in the presence of AsA. Meanwhile, AsA treatment reduced the alterations of G1/S phase cell cycle regulation genes. These results suggest that oxidative stress-derived DNA damage still exists, although AsA treatment effectively reduces PAT toxicity. This implies that a combined condition is required for complete blockade of PAT toxicity.

Phytotoxicity Evaluation of Type B Trichothecenes Using a Chlamydomonas reinhardtii Model System

Type B trichothecenes, which consist of deoxynivalenol (DON) and nivalenol (NIV) as the major end products, are produced by phytotoxic fungi, such as the Fusarium species, and pollute arable fields across the world. The DON toxicity has been investigated using various types of cell systems or animal bioassays. The evaluation of NIV toxicity, however, has been relatively restricted because of its lower level compared with DON. In this study, the Chlamydomonas reinhardtii testing system, which has been reported to have adequate NIV sensitivity, was reinvestigated under different mycotoxin concentrations and light conditions. The best concentration of DON and NIV, and their derivatives, for test conditions was found to be 25 ppm (2.5 × 10−2 mg/mL). In all light test conditions, DON, NIV, and fusarenon-X (FusX) indicated significant growth inhibition regardless of whether a light source existed, or under differential wavelength conditions. FusX growth was also influenced by changes in photon flux density. These results suggest that C. reinhardtii is an appropriate evaluation system for type B trichothecenes.

Low Toxicity of Deoxynivalenol-3-Glucoside in Microbial Cells

Host plants excrete a glucosylation enzyme onto the plant surface that changes mycotoxins derived from fungal secondary metabolites to glucosylated products. Deoxynivalenol-3-glucoside (DON3G) is synthesized by grain uridine diphosphate-glucosyltransferase, and is found worldwide, although information on its toxicity is lacking. Here, we conducted growth tests and DNA microarray analysis to elucidate the characteristics of DON3G. The Saccharomyces cerevisiae PDR5 mutant strain exposed to DON3G demonstrated similar growth to the dimethyl sulfoxide control, and DNA microarray analysis revealed limited differences. Only 10 genes were extracted, and the expression profile of stress response genes was similar to that of DON, in contrast to metabolism genes like SER3, which encodes 3-phosphoglycerate dehydrogenase. Growth tests with Chlamydomonas reinhardtii also showed a similar growth rate to the control sample. These results suggest that DON3G has extremely low toxicity to these cells, and the glucosylation of mycotoxins is a useful protective mechanism not only for host plants, but also for other species.

Regulation of metabolic products and gene expression in Fusarium asiaticum by agmatine addition

The metabolic products resulting from the cultivation of F. asiaticum in agmatine were identified using capillary electrophoresis–time of flight mass spectrometry. Glyoxylic acid was detected from fungal cultures grown in agmatine, while it was absent in control cells. The abundance of other metabolic products of the glycolytic pathway also increased because of agmatine; however, there was no increase in the amounts of pyruvic acid or metabolites from the tricarboxylic acid cycle. Moreover, gene expression levels within Fusarium asiaticum exposed to agmatine were analyzed by DNA microarray. Changes in gene expression levels directed the changes in metabolic products. Our results suggest that acetyl coenzyme A, which is a starting substrate for the biosynthesis of deoxynivalenol (DON), was simultaneously produced by activated β-oxidation. Furthermore, the content of 4-aminobutyrate (GABA) was increased in the agmatine addition culture medium. GABA can be synthesized from agmatine through putrescine and might influence the regulation of DON-related genes.

Acetylated Deoxynivalenol Generates Differences of Gene Expression that Discriminate Trichothecene Toxicity

Deoxynivalenol (DON), which is a toxic secondary metabolite generated by Fusarium species, is synthesized through two separate acetylation pathways. Both acetylation derivatives, 3-acetyl-DON (3ADON) and 15-acetyl-DON (15ADON), also contaminate grain and corn widely. These derivatives are deacetylated via a variety of processes after ingestion, so it has been suggested that they have the same toxicity as DON. However, in the intestinal entry region such as the duodenum, the derivatives might come into contact with intestinal epithelium cells because metabolism by microflora or import into the body has not progressed. Therefore, the differences of toxicity between DON and these derivatives need to be investigated. Here, we observed gene expression changes in the yeast pdr5Δ mutant strain under concentration-dependent mycotoxin exposure conditions. 15ADON exposure induced significant gene expression changes and DON exposure generally had a similar but smaller effect. However, the glucose transporter genes HXT2 and HXT4 showed converse trends. 3ADON also induced a different expression trend in these genes than DON and 15ADON. These differences in gene expression suggest that DON and its derivatives have different effects on cells.

Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi

Aflatoxin (AF) is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon) was added to a culture medium containing cyclodextrin (CD) to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence.

RNA Preparation of Saccharomyces cerevisiae Using the Digestion Method may Give Misleading Results

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

Light-Irradiation Wavelength and Intensity Changes Influence Aflatoxin Synthesis in Fungi

Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused on aflatoxin (AF), which is a mycotoxin, and the types of light irradiation that increase AF synthesis. Light-irradiation tests using the visible region indicated that blue wavelengths in the lower 500 nm region promoted AF synthesis. In contrast, red wavelengths of 660 nm resulted in limited significant changes compared with dark conditions. Irradiation tests with different intensity levels indicated that a low light intensity increased AF synthesis. For one fungal strain, light irradiation decreased the AF synthesis under all wavelength conditions. However, the decrease was mitigated by 525 nm low intensity irradiation. Thus, blue-green low intensity irradiation may increase AF synthesis in fungi.