Immunological methods are now widely used for screening and quantification of mycotoxins in cereals and foods. While, in feeds, there are few reports on immunoassays for screening and no quantitative methods have been developed. These facts suggest that one of the general advantages of immunoassay, direct application to the crude extract, does not work in the case of feed samples. This means that too much impurities that interfere the immunoreactions are contained in feeds and some clean up steps will be required for immunoassay for feeds. As of now, the immunoaffinity chromatography is probably most suitable for this purpose.
Aflatoxin B1 (AFB1)-induced hepatocellular carcinoma Kagura-2 cells overexpress c-myc oncogene, which has been implicated in both cell proliferation and apoptosis. To gain an insight into the molecular mechanism of AFB1 hepatocarcinogenesis, Kagura-2 cell death induced by an anticancer agent, sodium 5, 6-benzylideneascorbate (SBA), was studied. The dying cells showed typical characteristics of apoptosis such as nuclear fragmentation, chromosomal condensation and DNA fragmentation by internucleosomal cleavage. The apoptotic death was inhibited by the addition of cycloheximide (CHX), suggesting the requirement for new protein synthesis. However, the induction of Ca2+/Mg2+-dependent endonuclease activities and the alteration of c-myc gene expression in SBA-induced apoptosis were hardly detected. In addition, SBA-induced apoptosis was markedly suppressed by dexamethasone (DEX), insulin and P3 fraction, which was separated from the conditioned medium of Kagura-2 cells (K2CM) by Sephadex G-200 column chromatography and could stimulate Kagura-2 cell growth. P3 fraction also inhibited DNA fragmentation of Kagura-2 cells induced by serum deprivation. These results suggest that SBA induces apoptosis through the interference with the function of growth/survival factors acting in an autocrine and/or a paracrine mechanism or their signal transduction pathways. It is also possible that growth/survival factors play a critical role in the multistep hepatocarcinogenesis of AFB1 together with the deregulated expression of c-myc oncogene.
The effect of deoxynivalenol (DON) on Salmonella enteritidis infection was investigated in male Balb/c mice. Mice were given water containing 2 ppm DON or no toxin for 3 weeks. DON did not affect body weight and the spleen and liver weight. Mice were intubated with S. enteritidis (3×106 CFU) at 2 weeks of the experiment, and then enumeration of bacteria in the liver, spleen and mesentric lymph nodes (MLN) was examined for 1 week. Salmonellae increased more rapidly in the MLN and liver of the DON-treated mice than in those of the DON-untreated mice. Salmonella counts in the spleen were highter in the DON-treated mice than in untreated mice (p<0.05). These results indicate that drinking of 2 ppm DON reduces resistance to peroral infection of S. enteritidis, presumably by inhibiting the cell-mediated immune function.
The cytotoxicity of rubratoxin B was studied in human hepatoma cell HepG2, human uterine tumor cell HeLa and mouse ascites tumor cell Ehrlich. Exposure to rubratoxin B resulted in the cultured cells to be shrunk, and their volumes reduced. The inhibitory dose 50% (ID50) of the water soluble tetrazolium-1 assay (Wst-1; measurement of the activity of mitochondrial enzyme) were 2-to 3.6-fold of those of bromo-deoxyuridine incorporation (BrdU ; measurement of the activity of replication). Rubratoxin B did not affect the cell cycle, but brought about a dose-dependent increase of degraded chromosomal DNA. The reduction of cell volume and the degradation of chromosomal DNA may indicate that the toxicity of rubratoxin B is the consequence of apoptosis.
Novel monoclonal antibodies (mAb) against fumonisin B1 (FB1) were prepared and applied to an indirect competitive ELISA (ic-ELISA). BC6F1 female mice were immunized with a FB1-keyhole limpet hemocyanin conjugate (FB1-KLH). Trials of cell fusion yielded four hybridomas secreting mAb reactive with fumonisins (FB1-1, 2, 3, 4). FB1-2 mAb showing the highest reactivity to FB1 was used to construct an ic-ELISA. The detection limit was 0.2 ng FB1/ml in buffer solution. The relative cross-reactivities to FB2 and FB3 were 224% and 72% of FB1, respectively. Corn powder was firstly extracted with methanol-water (3 : 1), and the extract diluted with PBS (100 times) was directly applied to the ELISA. The recoveries of FB1 from corn spiked in range of 100-800 ppb FB1 were averaged to 88.4%. Corn samples (n=39) from China and Vietnam were analyzed for fumonisins by the present ic-ELISA, along with HPLC analysis, with a coefficient of correlation of 0.8. The detection limit of FB1 in this novel ELISA method combined with the one-step extraction procedure was 80 ppb, which is 10-60 fold lower than the ELISA previously repoted with anti-FB1 mAb.