Despite the numerous known beneficial effects of flavonoids, knowledge regarding their actions on hepatotoxicity is scanty. We therefore investigated whether the flavonoids quercetin and apigenin could protect human hepatoma cell line HuH-7 against rubratoxin B-induced toxicity. These flavonoids alone markedly retarded cell proliferation in a dose-dependent manner. Quercetin significantly lessened the inhibitory effects of rubratoxin B on cell proliferation at all concentrations tested (30-100 μM), however apigenin was protective only at low concentration (9 μM). Rubratoxin B stimulated the secretion of tumor necrosis factor- α and macrophage colony stimulating factor, and quercetin and apigenin dose-dependently inhibited the rubratoxin B-induced secretion of these cytokines. Apigenin appears to be a more potent inhibitor of cytokine secretion than is quercetin. Together our results suggest that quercetin and apigenin are protective against rubratoxin B-induced cytotoxicity.
We previously reported that 14-3-3 β mRNA is overexpressed as well as c-myc mRNA in aflatoxin B1 (AFB1)-induced rat hepatoma K2 cells and 14-3-3 β plays a crucial role in abnormal growth of K2 cells. The 14-3-3 family proteins are involved in important cellular processes such as signal transduction, cell cycle control, apoptosis and malignant transformation. The 14-3-3 proteins bind to phosphorylated form of proteins and a large number of their binding partners have been reported. Here, we isolated a novel binding partner of 14-3-3 β by yeast two-hybrid screening using 14-3-3 β as a bait, and it was termed fourteen-three-three beta interactant 1(FBI1). FBI1 mRNA was strongly expressed in K2 cells as compared with the normal rat liver tissue. Furthermore expression of antisense FBI1 RNA significantly suppressed anchorage-indepandent growth of K2 cells as a hallmark of tumor cells in vitro. These results suggest that FBI1 implicates in abnormal growth of K2 cells through the cooperation with 14-3-3 β.
Aflatoxin B1 (AFB1) is known to be one of the most potent hepatocarcinogens and causes hepatocellular carcinomas in various experimental animals. We have previously reported that AFB1-induced rat hepatocellular carcinoma K2 cells over-express 14-3-3 β and c-myc genes which are the key regulators of in vitro and in vivo growth of K2 cells. Nonetheless, it has been speculated that in addition to these genes, some other genes also implicate in AFB1 hepatocarcinogenesis, because AFB1 induces genomic alterations. Here, we report the cloning of cDNA encoding rat nuclear protein NC33 (nuclear coiled-coil protein 33 kDa) as a new candidate responsible for AFB1 hepatocarcinogenesis. The expression level of NC33 mRNA in K2 cells was extremely higher than that in normal rat liver. The colony forming ability of K2 cells in semi-solid medium as a hallmark of tumor cells in vitro was diminished by the down-regulation of NC33 gene expression. Thus, it is most likely that the deregulated expression of NC33 gene is involved in AFB1 hepatocarcinogenesis.
The DNA microarray technology is a very useful tool for a comprehensive analysis of the gene expression profile. On the other hand, cost, sensitivity, accuracy, and reproducibility are some of the many problems that must still be solved for this technology to be widely used. The performance of DNA microarray system such as accuracy and reproducibility is mainly defined by the fidelity of probe DNA for the abundance of cellular RNA by or the hybridization efficiency that the labeled target DNA hybridizes to probe DNA. In this study, we manufactured a highly sensitive oligonucleotide microarray system in order to analyze expression profiles comprehensively. In the conventional immobilized hybridization system, oligonucleotide probes which have high sequence specificities could not hybridize to probe DNA uniformly, and they brought nonuniform hybridization depended on fluorescent dyes. So, in order to reduce the ununiformity of hybridization efficiency, we attempted rotated hybridization system using chamber that the hybridization solution can circulate during the reaction. The rotary hybridization reduces uniformity of hybridization efficiency and results in the reduction of the fluorescent dye bias caused by the different amount of hybridized probe between differently labeled DNA. Moreover, by comparing with the expression profiles obtained in quantitative RT-PCR, we showed that the accuracy of the provided data by rotary hybridization was significantly superior to those by immobilized hybridization. We achieved highly accurate and reproducible DNA microarray analysis system using oligonucleotide microarray system.
All the aflatoxigenic fungi belong to genus Aspergillus section Flavi, and they are very close each other morphologically and genetically. The fungi sometime should be identified in combination of the morphological and molecular techniques in addition of mycotoxin production. A new aflatoxigenic species, A. nomius, is supported by cytochrome b gene analysis and hetero duplex analysis. The fungus is often found in sugarcane fields, especially of the southernmost islands, Japan. A. flavus is more aggressive than A. parasiticus to corn, rice and other crops whereas A. parasiticus infects peanut fruits more often than other crops. Aflatoxin contamination is influenced by a variety of environmental factors including climate conditions and biological factors such as susceptibility and occurrence of vector because the toxigenic fungi are weak “pathogen”. Drought stress prior to the harvest become the plant more susceptible to the fungi in corn and peanuts. So, preharvest control of the fungi is also important to reduce the contamination. Sterigmatocystin contamination by Aspergillus versicolor may occur when foodstuffs are kept for long time.
Ochratoxin A (OTA) was discovered by South African scientists during screening for toxigenic fungi that were belonging to strains of Aspergillus ochraceus. In contrast to the discovery of the aflatoxins, which was promoted by Turkey X disease, OTA has been treated as unimportant at that time. However, OTA became increasingly more interested in after discovery of natural occurrence of OTA in corn in 1969. OTA is a nephrotoxic and carcinogenic mycotoxin produced by Penicillium verrucosum in temperate or cold climate and a number of species of Aspergillus in tropical and subtropical area of the world, and is known to occur throughout the many kind of foods such as cereal, beans, coffee, cocoa, dried fruits, wine, beer, and spices. OTA has a half-life of about 35 days in humans, and was detected from human blood and breast milk, indicating a permanent dietary exposure. Under these circumstances, recently, the importance of OTA was more and more reflected by the works on risk assessment by the international organization such as the Joint FAO/WHO Expert Committee on Food Additives and European Union. In this paper, several surveillances of natural occurrence of OTA in foods by the international organizations and several countries are described. The carcinogenicity and genotoxicity of OTA are also summarized.